Issue w/ running cbSeurat: argument "counts" is missing, with no default

maximilianh / cellBrowser

main repo: https://github.com/ucscGenomeBrowser/cellBrowser/ - Python pipeline and Javascript scatter plot library for single-cell datasets, http://cellbrowser.rtfd.org

https://github.com/ucscGenomeBrowser/cellBrowser/

GNU General Public License v3.0

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Issue w/ running cbSeurat: argument "counts" is missing, with no default #265

Open bugacov opened 10 months ago

bugacov commented 10 months ago

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes = 200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

maximilianh commented 10 months ago

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes = 200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

— Reply to this email directly, view it on GitHub https://github.com/maximilianh/cellBrowser/issues/265, or unsubscribe https://github.com/notifications/unsubscribe-auth/AACL4TO4GXYNLDOX6XXN723YHDG7NAVCNFSM6AAAAABABU2KUWVHI2DSMVQWIX3LMV43ASLTON2WKOZSGAYTSMRYHEYDEMQ . You are receiving this because you are subscribed to this thread.Message ID: @.***>

bugacov commented 10 months ago

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes = 200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

— Reply to this email directly, view it on GitHub https://github.com/maximilianh/cellBrowser/issues/265 https://urldefense.us/v2/url?u=https-3A__github.com_maximilianh_cellBrowser_issues_265-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Rn_d-dYi9hOCejUz6BmvEpt67tZKG5RdMmhzO9DMIuTT3Wwjee1-4ShYtAtTFlQC&s=_CNzKLGxvMTMEzPRUkV2wqF-m-UbIr5pxvomm4lTp5U&e=, or unsubscribe https://github.com/notifications/unsubscribe-auth/AACL4TO4GXYNLDOX6XXN723YHDG7NAVCNFSM6AAAAABABU2KUWVHI2DSMVQWIX3LMV43ASLTON2WKOZSGAYTSMRYHEYDEMQ https://urldefense.us/v2/url?u=https-3A__github.com_notifications_unsubscribe-2Dauth_AACL4TO4GXYNLDOX6XXN723YHDG7NAVCNFSM6AAAAABABU2KUWVHI2DSMVQWIX3LMV43ASLTON2WKOZSGAYTSMRYHEYDEMQ-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Rn_d-dYi9hOCejUz6BmvEpt67tZKG5RdMmhzO9DMIuTT3Wwjee1-4ShYtAtTFlQC&s=Q59mfO72ssXd-s1xrxGwuQuGf3vdTn2Fp7qZdygMWNs&e= . You are receiving this because you are subscribed to this thread.Message ID: @.***>

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maximilianh commented 10 months ago

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes = 200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

— Reply to this email directly, view it on GitHub https://github.com/maximilianh/cellBrowser/issues/265< https://urldefense.us/v2/url?u=https-3A__github.com_maximilianh_cellBrowser_issues_265-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Rn_d-dYi9hOCejUz6BmvEpt67tZKG5RdMmhzO9DMIuTT3Wwjee1-4ShYtAtTFlQC&s=_CNzKLGxvMTMEzPRUkV2wqF-m-UbIr5pxvomm4lTp5U&e=>, or unsubscribe < https://github.com/notifications/unsubscribe-auth/AACL4TO4GXYNLDOX6XXN723YHDG7NAVCNFSM6AAAAABABU2KUWVHI2DSMVQWIX3LMV43ASLTON2WKOZSGAYTSMRYHEYDEMQ < https://urldefense.us/v2/url?u=https-3A__github.com_notifications_unsubscribe-2Dauth_AACL4TO4GXYNLDOX6XXN723YHDG7NAVCNFSM6AAAAABABU2KUWVHI2DSMVQWIX3LMV43ASLTON2WKOZSGAYTSMRYHEYDEMQ-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Rn_d-dYi9hOCejUz6BmvEpt67tZKG5RdMmhzO9DMIuTT3Wwjee1-4ShYtAtTFlQC&s=Q59mfO72ssXd-s1xrxGwuQuGf3vdTn2Fp7qZdygMWNs&e=>

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bugacov commented 10 months ago

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes = 200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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maximilianh commented 10 months ago

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes = 200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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bugacov commented 10 months ago

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes = 200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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maximilianh commented 10 months ago

Yes, the error is because your filenames don't follow the usual convention. Can you rename your files to matrix.mtx.gz and features.tsv.gz and barcodes.tsv.gz ? Or just create symlinks with this name. Then run it again.

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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bugacov commented 10 months ago

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData] ls -l test1/ total 106608 -rw-r--r--. 1 root root 46082 Dec 4 19:17 barcodes.tsv.gz -rw-r--r--. 1 root root 251118 Dec 4 19:17 features.tsv.gz -rw-r--r--. 1 root root 108861485 Dec 4 18:51 matrix.mtx.gz

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

ValueError: Reading with filekey 'test1' failed, the inferred filename PosixPath('write/test1.h5ad') does not exist. If you intended to provide a filename, either use a filename ending on one of the available extensions {'h5', 'anndata', 'txt', 'data', 'mtx.gz', 'tab', 'xlsx', 'tsv', 'h5ad', 'csv', 'mtx', 'loom', 'soft.gz'} or pass the parameter ext.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Monday, December 4, 2023 at 11:15 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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bugacov commented 10 months ago

Hi….I tried to run it on the example files from Scanpy example given here https://scanpy-tutorials.readthedocs.io/en/latest/pbmc3k.html

wget http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz -O data/pbmc3k_filtered_gene_bc_matrices.tar.gzhttp://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz

and I cannot get cbScanpy to run on them.

I built the .h5ad file in python using

import numpy as np import pandas as pd import scanpy as sc

adata = sc.read_10x_mtx( 'data/filtered_gene_bc_matrices/hg19/', # the directory with the .mtx file var_names='gene_symbols', # use gene symbols for the variable names (variables-axis index) cache=True) # write a cache file for faster subsequent reading

adata

and then tried to run the

cbImportScanpy -i filtered_gene_bc_matrices-hg19-matrix.h5ad -o pbmc3kImportScanpy

But that give errors too.

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 11:36 AM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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bugacov commented 10 months ago

I actually uninstall cellbrowser and installed it again and now is doing a bit more but still throwing errors:

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data] cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Loading Scanpy libraries INFO:root:Creating scanpyOut INFO:root:Loading Scanpy libraries INFO:root:cbScanpy $Id$ INFO:root:Command: /usr/local/bin/cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Matrix input file: filtered_gene_bc_matrices/hg19/matrix.mtx INFO:root:Restricting OPENBLAS to 4 threads INFO:root:Start time: 2023-12-05 04:30:26.199478

anndata 0.10.3 scanpy 1.9.6

PIL 10.0.0 asciitree NA cellbrowser v1.2.3 cffi 1.14.5 colorama 0.4.4 cycler 0.12.1 cython_runtime NA dateutil 2.8.2 entrypoints 0.4 exceptiongroup 1.2.0 fasteners 0.18 get_annotations NA h5py 3.10.0 igraph 0.10.8 importlib_resources NA joblib 1.3.2 kiwisolver 1.4.5 llvmlite 0.41.1 louvain 0.8.1 matplotlib 3.8.2 mpl_toolkits NA natsort 8.4.0 numba 0.58.1 numcodecs 0.11.0 numpy 1.25.2 packaging 23.1 pandas 2.1.3 pkg_resources NA psutil 5.8.0 pyparsing 3.1.1 pytz 2021.1 scipy 1.11.3 session_info 1.0.0 setuptools_scm NA six 1.15.0 sklearn 1.3.2 texttable 1.7.0 threadpoolctl 3.2.0 typing_extensions NA yaml 5.4.1 zarr 2.16.1 zipp NA zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)] Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-05 04:30 INFO:root:Loading Scanpy libraries INFO:root:Loading expression matrix: mtx format INFO:root:Data has 2700 samples/observations INFO:root:Data has 32738 genes/variables INFO:root:Basic filtering: keep only cells with min 200 genes INFO:root:Basic filtering: keep only gene with min 3 cells INFO:root:After filtering: Data has 2700 samples/observations and 13714 genes/variables INFO:root:Making gene names unique INFO:root:'geneIdType' is not specified in config file or set to 'auto'. INFO:root:Auto-detected gene IDs type: symbols INFO:root:Remove cells with more than 0.050000 percent of mitochondrial genes INFO:root:Computing percentage of mitochondrial genes WARNING: saving figure to file scanpyOut/figs/violin.png INFO:root:Remove cells with less than 10 and more than 15000 genes INFO:root:Keeping only cells with < 15000 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Keeping only cells with > 10 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Expression normalization, counts per cell = 10000 INFO:root:Did log2'ing of data INFO:root:Finding highly variable genes WARNING: saving figure to file scanpyOut/figs/filter_genes_dispersion.png INFO:root:After high-var filtering: Data has 2643 samples/observations and 1844 genes/variables INFO:root:Regressing out percent_mito and number of UMIs INFO:root:Scaling data, max_value=10 INFO:root:Performing initial PCA, number of PCs: 100 WARNING: saving figure to file scanpyOut/figs/pca_variance_ratio.png INFO:root:Estimating number of useful PCs based on Shekar et al, Cell 2016 INFO:root:PC weight cutoff used is (sqrt(# of Genes/# of cells) + 1)^2 INFO:root:See http://www.cell.com/cell/fulltext/S0092-8674(16)31007-8, STAR methods INFO:root:7 PCs will be used for tSNE and clustering INFO:root:Performing tSNE INFO:root:Running knn, using 7 PCs and 6 neighbors INFO:root:Performing Louvain Clustering, resolution 1.000000 INFO:root:Found 13 louvain clusters INFO:root:Using cluster annotation from field: louvain WARNING: saving figure to file scanpyOut/figs/tsne.png INFO:root:Performing UMAP INFO:root:Performing Layout 'drl' = DrL Distributed Recursive Layout INFO:root:Finding top markers for each cluster INFO:root:Loading Scanpy libraries Traceback (most recent call last): File "/usr/local/bin/cbScanpy", line 8, in sys.exit(cbScanpyCli()) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6643, in cbScanpyCli adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname, skipMarkers) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6432, in cbScanpy adata = runSafeRankGenesGroups(adata, clusterField, minCells=5) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4651, in runSafeRankGenesGroups clusterCellCounts = list(adata.obs.groupby([clusterField]).apply(len).iteritems()) File "/usr/local/lib64/python3.9/site-packages/pandas/core/generic.py", line 6204, in getattr return object.getattribute(self, name) AttributeError: 'Series' object has no attribute 'iteritems' facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data]

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 7:49 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hi….I tried to run it on the example files from Scanpy example given here https://scanpy-tutorials.readthedocs.io/en/latest/pbmc3k.html

and I cannot get cbScanpy to run on them.

I built the .h5ad file in python using

import numpy as np import pandas as pd import scanpy as sc

adata

and then tried to run the

cbImportScanpy -i filtered_gene_bc_matrices-hg19-matrix.h5ad -o pbmc3kImportScanpy

But that give errors too.

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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maximilianh commented 10 months ago

Great, so this almost worked, it's only stuck at the markers calculation step at the end now. This should be easy to fix.

You're using Python 3.9 from 2020 and combine that with the most recent scanpy version, just a few weeks old. We didn't test this combination. You could downgrade scanpy to a few versions older. But yes, it should normally work, it's possible I have to add yet another workaround (I've kept adding workarounds for the different scanpy versions, every few months, something is breaking there).

As soon as I can reproduce this error, I can fix it. Can you tell me where you downloaded the mtx file from? Is this your own file or a 10X pbmc demo file?

On Tue, Dec 5, 2023 at 5:33 AM bugacov @.***> wrote:

I actually uninstall cellbrowser and installed it again and now is doing a bit more but still throwing errors:

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data] cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Loading Scanpy libraries INFO:root:Creating scanpyOut INFO:root:Loading Scanpy libraries INFO:root:cbScanpy $Id$ INFO:root:Command: /usr/local/bin/cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Matrix input file: filtered_gene_bc_matrices/hg19/matrix.mtx INFO:root:Restricting OPENBLAS to 4 threads INFO:root:Start time: 2023-12-05 04:30:26.199478

anndata 0.10.3 scanpy 1.9.6

PIL 10.0.0 asciitree NA cellbrowser v1.2.3 cffi 1.14.5 colorama 0.4.4 cycler 0.12.1 cython_runtime NA dateutil 2.8.2 entrypoints 0.4 exceptiongroup 1.2.0 fasteners 0.18 get_annotations NA h5py 3.10.0 igraph 0.10.8 importlib_resources NA joblib 1.3.2 kiwisolver 1.4.5 llvmlite 0.41.1 louvain 0.8.1 matplotlib 3.8.2 mpl_toolkits NA natsort 8.4.0 numba 0.58.1 numcodecs 0.11.0 numpy 1.25.2 packaging 23.1 pandas 2.1.3 pkg_resources NA psutil 5.8.0 pyparsing 3.1.1 pytz 2021.1 scipy 1.11.3 session_info 1.0.0 setuptools_scm NA six 1.15.0 sklearn 1.3.2 texttable 1.7.0 threadpoolctl 3.2.0 typing_extensions NA yaml 5.4.1 zarr 2.16.1 zipp NA zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)] Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-05 04:30 INFO:root:Loading Scanpy libraries INFO:root:Loading expression matrix: mtx format INFO:root:Data has 2700 samples/observations INFO:root:Data has 32738 genes/variables INFO:root:Basic filtering: keep only cells with min 200 genes INFO:root:Basic filtering: keep only gene with min 3 cells INFO:root:After filtering: Data has 2700 samples/observations and 13714 genes/variables INFO:root:Making gene names unique INFO:root:'geneIdType' is not specified in config file or set to 'auto'. INFO:root:Auto-detected gene IDs type: symbols INFO:root:Remove cells with more than 0.050000 percent of mitochondrial genes INFO:root:Computing percentage of mitochondrial genes WARNING: saving figure to file scanpyOut/figs/violin.png INFO:root:Remove cells with less than 10 and more than 15000 genes INFO:root:Keeping only cells with < 15000 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Keeping only cells with > 10 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Expression normalization, counts per cell = 10000 INFO:root:Did log2'ing of data INFO:root:Finding highly variable genes WARNING: saving figure to file scanpyOut/figs/filter_genes_dispersion.png INFO:root:After high-var filtering: Data has 2643 samples/observations and 1844 genes/variables INFO:root:Regressing out percent_mito and number of UMIs INFO:root:Scaling data, max_value=10 INFO:root:Performing initial PCA, number of PCs: 100 WARNING: saving figure to file scanpyOut/figs/pca_variance_ratio.png INFO:root:Estimating number of useful PCs based on Shekar et al, Cell 2016 INFO:root:PC weight cutoff used is (sqrt(# of Genes/# of cells) + 1)^2 INFO:root:See http://www.cell.com/cell/fulltext/S0092-8674(16)31007-8, STAR methods INFO:root:7 PCs will be used for tSNE and clustering INFO:root:Performing tSNE INFO:root:Running knn, using 7 PCs and 6 neighbors INFO:root:Performing Louvain Clustering, resolution 1.000000 INFO:root:Found 13 louvain clusters INFO:root:Using cluster annotation from field: louvain WARNING: saving figure to file scanpyOut/figs/tsne.png INFO:root:Performing UMAP INFO:root:Performing Layout 'drl' = DrL Distributed Recursive Layout INFO:root:Finding top markers for each cluster INFO:root:Loading Scanpy libraries Traceback (most recent call last): File "/usr/local/bin/cbScanpy", line 8, in sys.exit(cbScanpyCli()) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6643, in cbScanpyCli adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname, skipMarkers) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6432, in cbScanpy adata = runSafeRankGenesGroups(adata, clusterField, minCells=5) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4651, in runSafeRankGenesGroups clusterCellCounts = list(adata.obs.groupby([clusterField]).apply(len).iteritems()) File "/usr/local/lib64/python3.9/site-packages/pandas/core/generic.py", line 6204, in getattr return object.getattribute(self, name) AttributeError: 'Series' object has no attribute 'iteritems' facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data]

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 7:49 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hi….I tried to run it on the example files from Scanpy example given here https://scanpy-tutorials.readthedocs.io/en/latest/pbmc3k.html

wget http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz -O data/pbmc3k_filtered_gene_bc_matrices.tar.gz< http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz>

and I cannot get cbScanpy to run on them.

I built the .h5ad file in python using

import numpy as np import pandas as pd import scanpy as sc

adata = sc.read_10x_mtx( 'data/filtered_gene_bc_matrices/hg19/', # the directory with the .mtx file var_names='gene_symbols', # use gene symbols for the variable names (variables-axis index) cache=True) # write a cache file for faster subsequent reading

adata

and then tried to run the

cbImportScanpy -i filtered_gene_bc_matrices-hg19-matrix.h5ad -o pbmc3kImportScanpy

But that give errors too.

I

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 11:36 AM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData] ls -l test1/ total 106608 -rw-r--r--. 1 root root 46082 Dec 4 19:17 barcodes.tsv.gz -rw-r--r--. 1 root root 251118 Dec 4 19:17 features.tsv.gz -rw-r--r--. 1 root root 108861485 Dec 4 18:51 matrix.mtx.gz

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

ValueError: Reading with filekey 'test1' failed, the inferred filename PosixPath('write/test1.h5ad') does not exist. If you intended to provide a filename, either use a filename ending on one of the available extensions {'h5', 'anndata', 'txt', 'data', 'mtx.gz', 'tab', 'xlsx', 'tsv', 'h5ad', 'csv', 'mtx', 'loom', 'soft.gz'} or pass the parameter ext.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Monday, December 4, 2023 at 11:15 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes, the error is because your filenames don't follow the usual convention. Can you rename your files to matrix.mtx.gz and features.tsv.gz and barcodes.tsv.gz ? Or just create symlinks with this name. Then run it again.

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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bugacov commented 10 months ago

It’t the pbmc demo file from the scanpy tutorial Thanks!!! Alejandro.

Sent from my iPhone

On Dec 5, 2023, at 1:15 AM, Maximilian Haeussler @.***> wrote:

Great, so this almost worked, it's only stuck at the markers calculation step at the end now. This should be easy to fix.

As soon as I can reproduce this error, I can fix it. Can you tell me where you downloaded the mtx file from? Is this your own file or a 10X pbmc demo file?

On Tue, Dec 5, 2023 at 5:33 AM bugacov @.***> wrote:

I actually uninstall cellbrowser and installed it again and now is doing a bit more but still throwing errors:

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data] cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Loading Scanpy libraries INFO:root:Creating scanpyOut INFO:root:Loading Scanpy libraries INFO:root:cbScanpy $Id$ INFO:root:Command: /usr/local/bin/cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Matrix input file: filtered_gene_bc_matrices/hg19/matrix.mtx INFO:root:Restricting OPENBLAS to 4 threads INFO:root:Start time: 2023-12-05 04:30:26.199478

anndata 0.10.3 scanpy 1.9.6

PIL 10.0.0 asciitree NA cellbrowser v1.2.3 cffi 1.14.5 colorama 0.4.4 cycler 0.12.1 cython_runtime NA dateutil 2.8.2 entrypoints 0.4 exceptiongroup 1.2.0 fasteners 0.18 get_annotations NA h5py 3.10.0 igraph 0.10.8 importlib_resources NA joblib 1.3.2 kiwisolver 1.4.5 llvmlite 0.41.1 louvain 0.8.1 matplotlib 3.8.2 mpl_toolkits NA natsort 8.4.0 numba 0.58.1 numcodecs 0.11.0 numpy 1.25.2 packaging 23.1 pandas 2.1.3 pkg_resources NA psutil 5.8.0 pyparsing 3.1.1 pytz 2021.1 scipy 1.11.3 session_info 1.0.0 setuptools_scm NA six 1.15.0 sklearn 1.3.2 texttable 1.7.0 threadpoolctl 3.2.0 typing_extensions NA yaml 5.4.1 zarr 2.16.1 zipp NA zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)] Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-05 04:30 INFO:root:Loading Scanpy libraries INFO:root:Loading expression matrix: mtx format INFO:root:Data has 2700 samples/observations INFO:root:Data has 32738 genes/variables INFO:root:Basic filtering: keep only cells with min 200 genes INFO:root:Basic filtering: keep only gene with min 3 cells INFO:root:After filtering: Data has 2700 samples/observations and 13714 genes/variables INFO:root:Making gene names unique INFO:root:'geneIdType' is not specified in config file or set to 'auto'. INFO:root:Auto-detected gene IDs type: symbols INFO:root:Remove cells with more than 0.050000 percent of mitochondrial genes INFO:root:Computing percentage of mitochondrial genes WARNING: saving figure to file scanpyOut/figs/violin.png INFO:root:Remove cells with less than 10 and more than 15000 genes INFO:root:Keeping only cells with < 15000 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Keeping only cells with > 10 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Expression normalization, counts per cell = 10000 INFO:root:Did log2'ing of data INFO:root:Finding highly variable genes WARNING: saving figure to file scanpyOut/figs/filter_genes_dispersion.png INFO:root:After high-var filtering: Data has 2643 samples/observations and 1844 genes/variables INFO:root:Regressing out percent_mito and number of UMIs INFO:root:Scaling data, max_value=10 INFO:root:Performing initial PCA, number of PCs: 100 WARNING: saving figure to file scanpyOut/figs/pca_variance_ratio.png INFO:root:Estimating number of useful PCs based on Shekar et al, Cell 2016 INFO:root:PC weight cutoff used is (sqrt(# of Genes/# of cells) + 1)^2 INFO:root:See http://www.cell.com/cell/fulltext/S0092-8674(16)31007-8 https://urldefense.us/v2/url?u=http-3A__www.cell.com_cell_fulltext_S0092-2D8674-2816-2931007-2D8&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=LRCerhXenZ3f2SWEPhr8kULBEGklU2NYVJ-O5nUQ-tA&e=, STAR methods INFO:root:7 PCs will be used for tSNE and clustering INFO:root:Performing tSNE INFO:root:Running knn, using 7 PCs and 6 neighbors INFO:root:Performing Louvain Clustering, resolution 1.000000 INFO:root:Found 13 louvain clusters INFO:root:Using cluster annotation from field: louvain WARNING: saving figure to file scanpyOut/figs/tsne.png INFO:root:Performing UMAP INFO:root:Performing Layout 'drl' = DrL Distributed Recursive Layout INFO:root:Finding top markers for each cluster INFO:root:Loading Scanpy libraries Traceback (most recent call last): File "/usr/local/bin/cbScanpy", line 8, in sys.exit(cbScanpyCli()) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6643, in cbScanpyCli adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname, skipMarkers) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6432, in cbScanpy adata = runSafeRankGenesGroups(adata, clusterField, minCells=5) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4651, in runSafeRankGenesGroups clusterCellCounts = list(adata.obs.groupby([clusterField]).apply(len).iteritems()) File "/usr/local/lib64/python3.9/site-packages/pandas/core/generic.py", line 6204, in getattr return object.getattribute(self, name) AttributeError: 'Series' object has no attribute 'iteritems' facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data]

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 7:49 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hi….I tried to run it on the example files from Scanpy example given here https://scanpy-tutorials.readthedocs.io/en/latest/pbmc3k.html https://urldefense.us/v2/url?u=https-3A__scanpy-2Dtutorials.readthedocs.io_en_latest_pbmc3k.html&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=fwTJ3jAfqNQubzg5eA4igcNVVQaoKw84DwvDYjI16tM&e=

wget http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e= -O data/pbmc3k_filtered_gene_bc_matrices.tar.gz< http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz>https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=

and I cannot get cbScanpy to run on them.

I built the .h5ad file in python using

import numpy as np import pandas as pd import scanpy as sc

adata = sc.read_10x_mtx( 'data/filtered_gene_bc_matrices/hg19/', # the directory with the .mtx file var_names='gene_symbols', # use gene symbols for the variable names (variables-axis index) cache=True) # write a cache file for faster subsequent reading

adata

and then tried to run the

cbImportScanpy -i filtered_gene_bc_matrices-hg19-matrix.h5ad -o pbmc3kImportScanpy

But that give errors too.

I

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 11:36 AM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData] ls -l test1/ total 106608 -rw-r--r--. 1 root root 46082 Dec 4 19:17 barcodes.tsv.gz -rw-r--r--. 1 root root 251118 Dec 4 19:17 features.tsv.gz -rw-r--r--. 1 root root 108861485 Dec 4 18:51 matrix.mtx.gz

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

ValueError: Reading with filekey 'test1' failed, the inferred filename PosixPath('write/test1.h5ad') does not exist. If you intended to provide a filename, either use a filename ending on one of the available extensions {'h5', 'anndata', 'txt', 'data', 'mtx.gz', 'tab', 'xlsx', 'tsv', 'h5ad', 'csv', 'mtx', 'loom', 'soft.gz'} or pass the parameter ext.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Monday, December 4, 2023 at 11:15 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes, the error is because your filenames don't follow the usual convention. Can you rename your files to matrix.mtx.gz and features.tsv.gz and barcodes.tsv.gz ? Or just create symlinks with this name. Then run it again.

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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bugacov commented 10 months ago

wget

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e= -O data/pbmc3k_filtered_gene_bc_matrices.tar.gz< http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz>https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=

Sent from my iPhone

On Dec 5, 2023, at 1:15 AM, Maximilian Haeussler @.***> wrote:

Great, so this almost worked, it's only stuck at the markers calculation step at the end now. This should be easy to fix.

As soon as I can reproduce this error, I can fix it. Can you tell me where you downloaded the mtx file from? Is this your own file or a 10X pbmc demo file?

On Tue, Dec 5, 2023 at 5:33 AM bugacov @.***> wrote:

I actually uninstall cellbrowser and installed it again and now is doing a bit more but still throwing errors:

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data] cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Loading Scanpy libraries INFO:root:Creating scanpyOut INFO:root:Loading Scanpy libraries INFO:root:cbScanpy $Id$ INFO:root:Command: /usr/local/bin/cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Matrix input file: filtered_gene_bc_matrices/hg19/matrix.mtx INFO:root:Restricting OPENBLAS to 4 threads INFO:root:Start time: 2023-12-05 04:30:26.199478

anndata 0.10.3 scanpy 1.9.6

PIL 10.0.0 asciitree NA cellbrowser v1.2.3 cffi 1.14.5 colorama 0.4.4 cycler 0.12.1 cython_runtime NA dateutil 2.8.2 entrypoints 0.4 exceptiongroup 1.2.0 fasteners 0.18 get_annotations NA h5py 3.10.0 igraph 0.10.8 importlib_resources NA joblib 1.3.2 kiwisolver 1.4.5 llvmlite 0.41.1 louvain 0.8.1 matplotlib 3.8.2 mpl_toolkits NA natsort 8.4.0 numba 0.58.1 numcodecs 0.11.0 numpy 1.25.2 packaging 23.1 pandas 2.1.3 pkg_resources NA psutil 5.8.0 pyparsing 3.1.1 pytz 2021.1 scipy 1.11.3 session_info 1.0.0 setuptools_scm NA six 1.15.0 sklearn 1.3.2 texttable 1.7.0 threadpoolctl 3.2.0 typing_extensions NA yaml 5.4.1 zarr 2.16.1 zipp NA zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)] Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-05 04:30 INFO:root:Loading Scanpy libraries INFO:root:Loading expression matrix: mtx format INFO:root:Data has 2700 samples/observations INFO:root:Data has 32738 genes/variables INFO:root:Basic filtering: keep only cells with min 200 genes INFO:root:Basic filtering: keep only gene with min 3 cells INFO:root:After filtering: Data has 2700 samples/observations and 13714 genes/variables INFO:root:Making gene names unique INFO:root:'geneIdType' is not specified in config file or set to 'auto'. INFO:root:Auto-detected gene IDs type: symbols INFO:root:Remove cells with more than 0.050000 percent of mitochondrial genes INFO:root:Computing percentage of mitochondrial genes WARNING: saving figure to file scanpyOut/figs/violin.png INFO:root:Remove cells with less than 10 and more than 15000 genes INFO:root:Keeping only cells with < 15000 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Keeping only cells with > 10 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Expression normalization, counts per cell = 10000 INFO:root:Did log2'ing of data INFO:root:Finding highly variable genes WARNING: saving figure to file scanpyOut/figs/filter_genes_dispersion.png INFO:root:After high-var filtering: Data has 2643 samples/observations and 1844 genes/variables INFO:root:Regressing out percent_mito and number of UMIs INFO:root:Scaling data, max_value=10 INFO:root:Performing initial PCA, number of PCs: 100 WARNING: saving figure to file scanpyOut/figs/pca_variance_ratio.png INFO:root:Estimating number of useful PCs based on Shekar et al, Cell 2016 INFO:root:PC weight cutoff used is (sqrt(# of Genes/# of cells) + 1)^2 INFO:root:See http://www.cell.com/cell/fulltext/S0092-8674(16)31007-8 https://urldefense.us/v2/url?u=http-3A__www.cell.com_cell_fulltext_S0092-2D8674-2816-2931007-2D8&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=LRCerhXenZ3f2SWEPhr8kULBEGklU2NYVJ-O5nUQ-tA&e=, STAR methods INFO:root:7 PCs will be used for tSNE and clustering INFO:root:Performing tSNE INFO:root:Running knn, using 7 PCs and 6 neighbors INFO:root:Performing Louvain Clustering, resolution 1.000000 INFO:root:Found 13 louvain clusters INFO:root:Using cluster annotation from field: louvain WARNING: saving figure to file scanpyOut/figs/tsne.png INFO:root:Performing UMAP INFO:root:Performing Layout 'drl' = DrL Distributed Recursive Layout INFO:root:Finding top markers for each cluster INFO:root:Loading Scanpy libraries Traceback (most recent call last): File "/usr/local/bin/cbScanpy", line 8, in sys.exit(cbScanpyCli()) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6643, in cbScanpyCli adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname, skipMarkers) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6432, in cbScanpy adata = runSafeRankGenesGroups(adata, clusterField, minCells=5) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4651, in runSafeRankGenesGroups clusterCellCounts = list(adata.obs.groupby([clusterField]).apply(len).iteritems()) File "/usr/local/lib64/python3.9/site-packages/pandas/core/generic.py", line 6204, in getattr return object.getattribute(self, name) AttributeError: 'Series' object has no attribute 'iteritems' facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data]

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 7:49 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hi….I tried to run it on the example files from Scanpy example given here https://scanpy-tutorials.readthedocs.io/en/latest/pbmc3k.html https://urldefense.us/v2/url?u=https-3A__scanpy-2Dtutorials.readthedocs.io_en_latest_pbmc3k.html&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=fwTJ3jAfqNQubzg5eA4igcNVVQaoKw84DwvDYjI16tM&e=

wget http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e= -O data/pbmc3k_filtered_gene_bc_matrices.tar.gz< http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz>https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=

and I cannot get cbScanpy to run on them.

I built the .h5ad file in python using

import numpy as np import pandas as pd import scanpy as sc

adata = sc.read_10x_mtx( 'data/filtered_gene_bc_matrices/hg19/', # the directory with the .mtx file var_names='gene_symbols', # use gene symbols for the variable names (variables-axis index) cache=True) # write a cache file for faster subsequent reading

adata

and then tried to run the

cbImportScanpy -i filtered_gene_bc_matrices-hg19-matrix.h5ad -o pbmc3kImportScanpy

But that give errors too.

I

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 11:36 AM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData] ls -l test1/ total 106608 -rw-r--r--. 1 root root 46082 Dec 4 19:17 barcodes.tsv.gz -rw-r--r--. 1 root root 251118 Dec 4 19:17 features.tsv.gz -rw-r--r--. 1 root root 108861485 Dec 4 18:51 matrix.mtx.gz

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

ValueError: Reading with filekey 'test1' failed, the inferred filename PosixPath('write/test1.h5ad') does not exist. If you intended to provide a filename, either use a filename ending on one of the available extensions {'h5', 'anndata', 'txt', 'data', 'mtx.gz', 'tab', 'xlsx', 'tsv', 'h5ad', 'csv', 'mtx', 'loom', 'soft.gz'} or pass the parameter ext.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Monday, December 4, 2023 at 11:15 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes, the error is because your filenames don't follow the usual convention. Can you rename your files to matrix.mtx.gz and features.tsv.gz and barcodes.tsv.gz ? Or just create symlinks with this name. Then run it again.

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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maximilianh commented 10 months ago

Ah, it's just that file. Yes, we do test and it works for me.

This looks a little like a problem with your scanpy installation... but we'll see.

I'm using Python 3.7 and Scanpy 1.9.2 and scipy 1.7.3

I can try to use the same versions that you have. Can you send me the list of these versions: Python, Scipy, Numpy and especially Scanpy.

On Tue, Dec 5, 2023 at 4:11 PM bugacov @.***> wrote:

wget

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e=>

-O data/pbmc3k_filtered_gene_bc_matrices.tar.gz<

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=>

Sent from my iPhone

On Dec 5, 2023, at 1:15 AM, Maximilian Haeussler @.***> wrote:

Great, so this almost worked, it's only stuck at the markers calculation step at the end now. This should be easy to fix.

You're using Python 3.9 from 2020 and combine that with the most recent scanpy version, just a few weeks old. We didn't test this combination. You could downgrade scanpy to a few versions older. But yes, it should normally work, it's possible I have to add yet another workaround (I've kept adding workarounds for the different scanpy versions, every few months, something is breaking there).

As soon as I can reproduce this error, I can fix it. Can you tell me where you downloaded the mtx file from? Is this your own file or a 10X pbmc demo file?

On Tue, Dec 5, 2023 at 5:33 AM bugacov @.***> wrote:

I actually uninstall cellbrowser and installed it again and now is doing a bit more but still throwing errors:

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data] cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Loading Scanpy libraries INFO:root:Creating scanpyOut INFO:root:Loading Scanpy libraries INFO:root:cbScanpy $Id$ INFO:root:Command: /usr/local/bin/cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Matrix input file: filtered_gene_bc_matrices/hg19/matrix.mtx INFO:root:Restricting OPENBLAS to 4 threads INFO:root:Start time: 2023-12-05 04:30:26.199478

anndata 0.10.3 scanpy 1.9.6

PIL 10.0.0 asciitree NA cellbrowser v1.2.3 cffi 1.14.5 colorama 0.4.4 cycler 0.12.1 cython_runtime NA dateutil 2.8.2 entrypoints 0.4 exceptiongroup 1.2.0 fasteners 0.18 get_annotations NA h5py 3.10.0 igraph 0.10.8 importlib_resources NA joblib 1.3.2 kiwisolver 1.4.5 llvmlite 0.41.1 louvain 0.8.1 matplotlib 3.8.2 mpl_toolkits NA natsort 8.4.0 numba 0.58.1 numcodecs 0.11.0 numpy 1.25.2 packaging 23.1 pandas 2.1.3 pkg_resources NA psutil 5.8.0 pyparsing 3.1.1 pytz 2021.1 scipy 1.11.3 session_info 1.0.0 setuptools_scm NA six 1.15.0 sklearn 1.3.2 texttable 1.7.0 threadpoolctl 3.2.0 typing_extensions NA yaml 5.4.1 zarr 2.16.1 zipp NA zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)] Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-05 04:30 INFO:root:Loading Scanpy libraries INFO:root:Loading expression matrix: mtx format INFO:root:Data has 2700 samples/observations INFO:root:Data has 32738 genes/variables INFO:root:Basic filtering: keep only cells with min 200 genes INFO:root:Basic filtering: keep only gene with min 3 cells INFO:root:After filtering: Data has 2700 samples/observations and 13714 genes/variables INFO:root:Making gene names unique INFO:root:'geneIdType' is not specified in config file or set to 'auto'. INFO:root:Auto-detected gene IDs type: symbols INFO:root:Remove cells with more than 0.050000 percent of mitochondrial genes INFO:root:Computing percentage of mitochondrial genes WARNING: saving figure to file scanpyOut/figs/violin.png INFO:root:Remove cells with less than 10 and more than 15000 genes INFO:root:Keeping only cells with < 15000 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Keeping only cells with > 10 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Expression normalization, counts per cell = 10000 INFO:root:Did log2'ing of data INFO:root:Finding highly variable genes WARNING: saving figure to file scanpyOut/figs/filter_genes_dispersion.png INFO:root:After high-var filtering: Data has 2643 samples/observations and 1844 genes/variables INFO:root:Regressing out percent_mito and number of UMIs INFO:root:Scaling data, max_value=10 INFO:root:Performing initial PCA, number of PCs: 100 WARNING: saving figure to file scanpyOut/figs/pca_variance_ratio.png INFO:root:Estimating number of useful PCs based on Shekar et al, Cell 2016 INFO:root:PC weight cutoff used is (sqrt(# of Genes/# of cells) + 1)^2 INFO:root:See http://www.cell.com/cell/fulltext/S0092-8674(16)31007-8< https://urldefense.us/v2/url?u=http-3A__www.cell.com_cell_fulltext_S0092-2D8674-2816-2931007-2D8&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=LRCerhXenZ3f2SWEPhr8kULBEGklU2NYVJ-O5nUQ-tA&e=>,

STAR methods INFO:root:7 PCs will be used for tSNE and clustering INFO:root:Performing tSNE INFO:root:Running knn, using 7 PCs and 6 neighbors INFO:root:Performing Louvain Clustering, resolution 1.000000 INFO:root:Found 13 louvain clusters INFO:root:Using cluster annotation from field: louvain WARNING: saving figure to file scanpyOut/figs/tsne.png INFO:root:Performing UMAP INFO:root:Performing Layout 'drl' = DrL Distributed Recursive Layout INFO:root:Finding top markers for each cluster INFO:root:Loading Scanpy libraries Traceback (most recent call last): File "/usr/local/bin/cbScanpy", line 8, in sys.exit(cbScanpyCli()) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6643, in cbScanpyCli adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname, skipMarkers) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6432, in cbScanpy adata = runSafeRankGenesGroups(adata, clusterField, minCells=5) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4651, in runSafeRankGenesGroups clusterCellCounts = list(adata.obs.groupby([clusterField]).apply(len).iteritems()) File "/usr/local/lib64/python3.9/site-packages/pandas/core/generic.py", line 6204, in getattr return object.getattribute(self, name) AttributeError: 'Series' object has no attribute 'iteritems' facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data]

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 7:49 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hi….I tried to run it on the example files from Scanpy example given here https://scanpy-tutorials.readthedocs.io/en/latest/pbmc3k.html< https://urldefense.us/v2/url?u=https-3A__scanpy-2Dtutorials.readthedocs.io_en_latest_pbmc3k.html&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=fwTJ3jAfqNQubzg5eA4igcNVVQaoKw84DwvDYjI16tM&e=>

wget

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e=>

-O data/pbmc3k_filtered_gene_bc_matrices.tar.gz<

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=>

and I cannot get cbScanpy to run on them.

I built the .h5ad file in python using

import numpy as np import pandas as pd import scanpy as sc

adata = sc.read_10x_mtx( 'data/filtered_gene_bc_matrices/hg19/', # the directory with the .mtx file var_names='gene_symbols', # use gene symbols for the variable names (variables-axis index) cache=True) # write a cache file for faster subsequent reading

adata

and then tried to run the

cbImportScanpy -i filtered_gene_bc_matrices-hg19-matrix.h5ad -o pbmc3kImportScanpy

But that give errors too.

I

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 11:36 AM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData] ls -l test1/ total 106608 -rw-r--r--. 1 root root 46082 Dec 4 19:17 barcodes.tsv.gz -rw-r--r--. 1 root root 251118 Dec 4 19:17 features.tsv.gz -rw-r--r--. 1 root root 108861485 Dec 4 18:51 matrix.mtx.gz

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

ValueError: Reading with filekey 'test1' failed, the inferred filename PosixPath('write/test1.h5ad') does not exist. If you intended to provide a filename, either use a filename ending on one of the available extensions {'h5', 'anndata', 'txt', 'data', 'mtx.gz', 'tab', 'xlsx', 'tsv', 'h5ad', 'csv', 'mtx', 'loom', 'soft.gz'} or pass the parameter ext.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Monday, December 4, 2023 at 11:15 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes, the error is because your filenames don't follow the usual convention. Can you rename your files to matrix.mtx.gz and features.tsv.gz and barcodes.tsv.gz ? Or just create symlinks with this name. Then run it again.

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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bugacov commented 10 months ago

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Tuesday, December 5, 2023 at 7:23 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Ah, it's just that file. Yes, we do test and it works for me.

This looks a little like a problem with your scanpy installation... but we'll see.

I'm using Python 3.7 and Scanpy 1.9.2 and scipy 1.7.3

I can try to use the same versions that you have. Can you send me the list of these versions: Python, Scipy, Numpy and especially Scanpy.

Python 3.9.18

scanpy==1.9.6

scipy==1.11.3

numpy==1.25.2

Thanks! Alejandro.

On Tue, Dec 5, 2023 at 4:11 PM bugacov @.***> wrote:

wget

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=vfqy0VZ_lZZYeWdNabrBSDEqpde9StRJR_FA1l6CILg&e= < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e=%3E

-O data/pbmc3k_filtered_gene_bc_matrices.tar.gz<

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=R827nUTL2RtYc_UutyP-51nWeHNYrD9Y0d0nnCisuhc&e= < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=%3E

Sent from my iPhone

On Dec 5, 2023, at 1:15 AM, Maximilian Haeussler @.***> wrote:

Great, so this almost worked, it's only stuck at the markers calculation step at the end now. This should be easy to fix.

You're using Python 3.9 from 2020 and combine that with the most recent scanpy version, just a few weeks old. We didn't test this combination. You could downgrade scanpy to a few versions older. But yes, it should normally work, it's possible I have to add yet another workaround (I've kept adding workarounds for the different scanpy versions, every few months, something is breaking there).

As soon as I can reproduce this error, I can fix it. Can you tell me where you downloaded the mtx file from? Is this your own file or a 10X pbmc demo file?

On Tue, Dec 5, 2023 at 5:33 AM bugacov @.***> wrote:

I actually uninstall cellbrowser and installed it again and now is doing a bit more but still throwing errors:

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data] cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Loading Scanpy libraries INFO:root:Creating scanpyOut INFO:root:Loading Scanpy libraries INFO:root:cbScanpy $Id$ INFO:root:Command: /usr/local/bin/cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Matrix input file: filtered_gene_bc_matrices/hg19/matrix.mtx INFO:root:Restricting OPENBLAS to 4 threads INFO:root:Start time: 2023-12-05 04:30:26.199478

anndata 0.10.3 scanpy 1.9.6

PIL 10.0.0 asciitree NA cellbrowser v1.2.3 cffi 1.14.5 colorama 0.4.4 cycler 0.12.1 cython_runtime NA dateutil 2.8.2 entrypoints 0.4 exceptiongroup 1.2.0 fasteners 0.18 get_annotations NA h5py 3.10.0 igraph 0.10.8 importlib_resources NA joblib 1.3.2 kiwisolver 1.4.5 llvmlite 0.41.1 louvain 0.8.1 matplotlib 3.8.2 mpl_toolkits NA natsort 8.4.0 numba 0.58.1 numcodecs 0.11.0 numpy 1.25.2 packaging 23.1 pandas 2.1.3 pkg_resources NA psutil 5.8.0 pyparsing 3.1.1 pytz 2021.1 scipy 1.11.3 session_info 1.0.0 setuptools_scm NA six 1.15.0 sklearn 1.3.2 texttable 1.7.0 threadpoolctl 3.2.0 typing_extensions NA yaml 5.4.1 zarr 2.16.1 zipp NA zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)] Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-05 04:30 INFO:root:Loading Scanpy libraries INFO:root:Loading expression matrix: mtx format INFO:root:Data has 2700 samples/observations INFO:root:Data has 32738 genes/variables INFO:root:Basic filtering: keep only cells with min 200 genes INFO:root:Basic filtering: keep only gene with min 3 cells INFO:root:After filtering: Data has 2700 samples/observations and 13714 genes/variables INFO:root:Making gene names unique INFO:root:'geneIdType' is not specified in config file or set to 'auto'. INFO:root:Auto-detected gene IDs type: symbols INFO:root:Remove cells with more than 0.050000 percent of mitochondrial genes INFO:root:Computing percentage of mitochondrial genes WARNING: saving figure to file scanpyOut/figs/violin.png INFO:root:Remove cells with less than 10 and more than 15000 genes INFO:root:Keeping only cells with < 15000 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Keeping only cells with > 10 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Expression normalization, counts per cell = 10000 INFO:root:Did log2'ing of data INFO:root:Finding highly variable genes WARNING: saving figure to file scanpyOut/figs/filter_genes_dispersion.png INFO:root:After high-var filtering: Data has 2643 samples/observations and 1844 genes/variables INFO:root:Regressing out percent_mito and number of UMIs INFO:root:Scaling data, max_value=10 INFO:root:Performing initial PCA, number of PCs: 100 WARNING: saving figure to file scanpyOut/figs/pca_variance_ratio.png INFO:root:Estimating number of useful PCs based on Shekar et al, Cell 2016 INFO:root:PC weight cutoff used is (sqrt(# of Genes/# of cells) + 1)^2 INFO:root:See http://www.cell.com/cell/fulltext/S0092-8674(16)31007-8<https://urldefense.us/v2/url?u=http-3A__www.cell.com_cell_fulltext_S0092-2D8674-2816-2931007-2D8-253C&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=V7ZgmTdvAWsVTOnGdosk-ZKl0Rd7rUdm2Pt_Q236vTI&e= https://urldefense.us/v2/url?u=http-3A__www.cell.com_cell_fulltext_S0092-2D8674-2816-2931007-2D8&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=LRCerhXenZ3f2SWEPhr8kULBEGklU2NYVJ-O5nUQ-tA&e=%3E,

STAR methods INFO:root:7 PCs will be used for tSNE and clustering INFO:root:Performing tSNE INFO:root:Running knn, using 7 PCs and 6 neighbors INFO:root:Performing Louvain Clustering, resolution 1.000000 INFO:root:Found 13 louvain clusters INFO:root:Using cluster annotation from field: louvain WARNING: saving figure to file scanpyOut/figs/tsne.png INFO:root:Performing UMAP INFO:root:Performing Layout 'drl' = DrL Distributed Recursive Layout INFO:root:Finding top markers for each cluster INFO:root:Loading Scanpy libraries Traceback (most recent call last): File "/usr/local/bin/cbScanpy", line 8, in sys.exit(cbScanpyCli()) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6643, in cbScanpyCli adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname, skipMarkers) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6432, in cbScanpy adata = runSafeRankGenesGroups(adata, clusterField, minCells=5) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4651, in runSafeRankGenesGroups clusterCellCounts = list(adata.obs.groupby([clusterField]).apply(len).iteritems()) File "/usr/local/lib64/python3.9/site-packages/pandas/core/generic.py", line 6204, in getattr return object.getattribute(self, name) AttributeError: 'Series' object has no attribute 'iteritems' facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data]

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 7:49 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hi….I tried to run it on the example files from Scanpy example given here https://scanpy-tutorials.readthedocs.io/en/latest/pbmc3k.html<https://urldefense.us/v2/url?u=https-3A__scanpy-2Dtutorials.readthedocs.io_en_latest_pbmc3k.html-253C&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=hkV8fjmu18Vu9JP99fI2Yr2oNegSCXgjEAuaMlSN-ps&e= https://urldefense.us/v2/url?u=https-3A__scanpy-2Dtutorials.readthedocs.io_en_latest_pbmc3k.html&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=fwTJ3jAfqNQubzg5eA4igcNVVQaoKw84DwvDYjI16tM&e=%3E

wget

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=vfqy0VZ_lZZYeWdNabrBSDEqpde9StRJR_FA1l6CILg&e= < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e=%3E

-O data/pbmc3k_filtered_gene_bc_matrices.tar.gz<

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=R827nUTL2RtYc_UutyP-51nWeHNYrD9Y0d0nnCisuhc&e= < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=%3E

and I cannot get cbScanpy to run on them.

I built the .h5ad file in python using

import numpy as np import pandas as pd import scanpy as sc

adata = sc.read_10x_mtx( 'data/filtered_gene_bc_matrices/hg19/', # the directory with the .mtx file var_names='gene_symbols', # use gene symbols for the variable names (variables-axis index) cache=True) # write a cache file for faster subsequent reading

adata

and then tried to run the

cbImportScanpy -i filtered_gene_bc_matrices-hg19-matrix.h5ad -o pbmc3kImportScanpy

But that give errors too.

I

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 11:36 AM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData] ls -l test1/ total 106608 -rw-r--r--. 1 root root 46082 Dec 4 19:17 barcodes.tsv.gz -rw-r--r--. 1 root root 251118 Dec 4 19:17 features.tsv.gz -rw-r--r--. 1 root root 108861485 Dec 4 18:51 matrix.mtx.gz

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

ValueError: Reading with filekey 'test1' failed, the inferred filename PosixPath('write/test1.h5ad') does not exist. If you intended to provide a filename, either use a filename ending on one of the available extensions {'h5', 'anndata', 'txt', 'data', 'mtx.gz', 'tab', 'xlsx', 'tsv', 'h5ad', 'csv', 'mtx', 'loom', 'soft.gz'} or pass the parameter ext.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Monday, December 4, 2023 at 11:15 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes, the error is because your filenames don't follow the usual convention. Can you rename your files to matrix.mtx.gz and features.tsv.gz and barcodes.tsv.gz ? Or just create symlinks with this name. Then run it again.

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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bugacov commented 9 months ago

Hi….I was wondering if you were able to reproduce my errors? I think that the main difference seems to be the Python version on my machine but I kinda constrained to stay on that version if possible. Let me know if there’s something else I can try.

Again the versions in my system are:

Python 3.9.18

scanpy==1.9.6

scipy==1.11.3

numpy==1.25.2

Thanks! Alejandro.

From: Alejandro Bugacov @.> Date: Tuesday, December 5, 2023 at 2:34 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Ah, it's just that file. Yes, we do test and it works for me.

This looks a little like a problem with your scanpy installation... but we'll see.

I'm using Python 3.7 and Scanpy 1.9.2 and scipy 1.7.3

I can try to use the same versions that you have. Can you send me the list of these versions: Python, Scipy, Numpy and especially Scanpy.

Python 3.9.18

scanpy==1.9.6

scipy==1.11.3

numpy==1.25.2

Thanks! Alejandro.

On Tue, Dec 5, 2023 at 4:11 PM bugacov @.***> wrote:

wget

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=vfqy0VZ_lZZYeWdNabrBSDEqpde9StRJR_FA1l6CILg&e= < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e=%3E

-O data/pbmc3k_filtered_gene_bc_matrices.tar.gz<

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=R827nUTL2RtYc_UutyP-51nWeHNYrD9Y0d0nnCisuhc&e= < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=%3E

Sent from my iPhone

On Dec 5, 2023, at 1:15 AM, Maximilian Haeussler @.***> wrote:

Great, so this almost worked, it's only stuck at the markers calculation step at the end now. This should be easy to fix.

You're using Python 3.9 from 2020 and combine that with the most recent scanpy version, just a few weeks old. We didn't test this combination. You could downgrade scanpy to a few versions older. But yes, it should normally work, it's possible I have to add yet another workaround (I've kept adding workarounds for the different scanpy versions, every few months, something is breaking there).

As soon as I can reproduce this error, I can fix it. Can you tell me where you downloaded the mtx file from? Is this your own file or a 10X pbmc demo file?

On Tue, Dec 5, 2023 at 5:33 AM bugacov @.***> wrote:

I actually uninstall cellbrowser and installed it again and now is doing a bit more but still throwing errors:

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data] cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Loading Scanpy libraries INFO:root:Creating scanpyOut INFO:root:Loading Scanpy libraries INFO:root:cbScanpy $Id$ INFO:root:Command: /usr/local/bin/cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Matrix input file: filtered_gene_bc_matrices/hg19/matrix.mtx INFO:root:Restricting OPENBLAS to 4 threads INFO:root:Start time: 2023-12-05 04:30:26.199478

anndata 0.10.3 scanpy 1.9.6

PIL 10.0.0 asciitree NA cellbrowser v1.2.3 cffi 1.14.5 colorama 0.4.4 cycler 0.12.1 cython_runtime NA dateutil 2.8.2 entrypoints 0.4 exceptiongroup 1.2.0 fasteners 0.18 get_annotations NA h5py 3.10.0 igraph 0.10.8 importlib_resources NA joblib 1.3.2 kiwisolver 1.4.5 llvmlite 0.41.1 louvain 0.8.1 matplotlib 3.8.2 mpl_toolkits NA natsort 8.4.0 numba 0.58.1 numcodecs 0.11.0 numpy 1.25.2 packaging 23.1 pandas 2.1.3 pkg_resources NA psutil 5.8.0 pyparsing 3.1.1 pytz 2021.1 scipy 1.11.3 session_info 1.0.0 setuptools_scm NA six 1.15.0 sklearn 1.3.2 texttable 1.7.0 threadpoolctl 3.2.0 typing_extensions NA yaml 5.4.1 zarr 2.16.1 zipp NA zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)] Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-05 04:30 INFO:root:Loading Scanpy libraries INFO:root:Loading expression matrix: mtx format INFO:root:Data has 2700 samples/observations INFO:root:Data has 32738 genes/variables INFO:root:Basic filtering: keep only cells with min 200 genes INFO:root:Basic filtering: keep only gene with min 3 cells INFO:root:After filtering: Data has 2700 samples/observations and 13714 genes/variables INFO:root:Making gene names unique INFO:root:'geneIdType' is not specified in config file or set to 'auto'. INFO:root:Auto-detected gene IDs type: symbols INFO:root:Remove cells with more than 0.050000 percent of mitochondrial genes INFO:root:Computing percentage of mitochondrial genes WARNING: saving figure to file scanpyOut/figs/violin.png INFO:root:Remove cells with less than 10 and more than 15000 genes INFO:root:Keeping only cells with < 15000 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Keeping only cells with > 10 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Expression normalization, counts per cell = 10000 INFO:root:Did log2'ing of data INFO:root:Finding highly variable genes WARNING: saving figure to file scanpyOut/figs/filter_genes_dispersion.png INFO:root:After high-var filtering: Data has 2643 samples/observations and 1844 genes/variables INFO:root:Regressing out percent_mito and number of UMIs INFO:root:Scaling data, max_value=10 INFO:root:Performing initial PCA, number of PCs: 100 WARNING: saving figure to file scanpyOut/figs/pca_variance_ratio.png INFO:root:Estimating number of useful PCs based on Shekar et al, Cell 2016 INFO:root:PC weight cutoff used is (sqrt(# of Genes/# of cells) + 1)^2 INFO:root:See http://www.cell.com/cell/fulltext/S0092-8674(16)31007-8<https://urldefense.us/v2/url?u=http-3A__www.cell.com_cell_fulltext_S0092-2D8674-2816-2931007-2D8-253C&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=V7ZgmTdvAWsVTOnGdosk-ZKl0Rd7rUdm2Pt_Q236vTI&e= https://urldefense.us/v2/url?u=http-3A__www.cell.com_cell_fulltext_S0092-2D8674-2816-2931007-2D8&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=LRCerhXenZ3f2SWEPhr8kULBEGklU2NYVJ-O5nUQ-tA&e=%3E,

STAR methods INFO:root:7 PCs will be used for tSNE and clustering INFO:root:Performing tSNE INFO:root:Running knn, using 7 PCs and 6 neighbors INFO:root:Performing Louvain Clustering, resolution 1.000000 INFO:root:Found 13 louvain clusters INFO:root:Using cluster annotation from field: louvain WARNING: saving figure to file scanpyOut/figs/tsne.png INFO:root:Performing UMAP INFO:root:Performing Layout 'drl' = DrL Distributed Recursive Layout INFO:root:Finding top markers for each cluster INFO:root:Loading Scanpy libraries Traceback (most recent call last): File "/usr/local/bin/cbScanpy", line 8, in sys.exit(cbScanpyCli()) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6643, in cbScanpyCli adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname, skipMarkers) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6432, in cbScanpy adata = runSafeRankGenesGroups(adata, clusterField, minCells=5) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4651, in runSafeRankGenesGroups clusterCellCounts = list(adata.obs.groupby([clusterField]).apply(len).iteritems()) File "/usr/local/lib64/python3.9/site-packages/pandas/core/generic.py", line 6204, in getattr return object.getattribute(self, name) AttributeError: 'Series' object has no attribute 'iteritems' facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data]

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 7:49 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hi….I tried to run it on the example files from Scanpy example given here https://scanpy-tutorials.readthedocs.io/en/latest/pbmc3k.html<https://urldefense.us/v2/url?u=https-3A__scanpy-2Dtutorials.readthedocs.io_en_latest_pbmc3k.html-253C&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=hkV8fjmu18Vu9JP99fI2Yr2oNegSCXgjEAuaMlSN-ps&e= https://urldefense.us/v2/url?u=https-3A__scanpy-2Dtutorials.readthedocs.io_en_latest_pbmc3k.html&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=fwTJ3jAfqNQubzg5eA4igcNVVQaoKw84DwvDYjI16tM&e=%3E

wget

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=vfqy0VZ_lZZYeWdNabrBSDEqpde9StRJR_FA1l6CILg&e= < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e=%3E

-O data/pbmc3k_filtered_gene_bc_matrices.tar.gz<

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=R827nUTL2RtYc_UutyP-51nWeHNYrD9Y0d0nnCisuhc&e= < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=%3E

and I cannot get cbScanpy to run on them.

I built the .h5ad file in python using

import numpy as np import pandas as pd import scanpy as sc

adata = sc.read_10x_mtx( 'data/filtered_gene_bc_matrices/hg19/', # the directory with the .mtx file var_names='gene_symbols', # use gene symbols for the variable names (variables-axis index) cache=True) # write a cache file for faster subsequent reading

adata

and then tried to run the

cbImportScanpy -i filtered_gene_bc_matrices-hg19-matrix.h5ad -o pbmc3kImportScanpy

But that give errors too.

I

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 11:36 AM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData] ls -l test1/ total 106608 -rw-r--r--. 1 root root 46082 Dec 4 19:17 barcodes.tsv.gz -rw-r--r--. 1 root root 251118 Dec 4 19:17 features.tsv.gz -rw-r--r--. 1 root root 108861485 Dec 4 18:51 matrix.mtx.gz

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

ValueError: Reading with filekey 'test1' failed, the inferred filename PosixPath('write/test1.h5ad') does not exist. If you intended to provide a filename, either use a filename ending on one of the available extensions {'h5', 'anndata', 'txt', 'data', 'mtx.gz', 'tab', 'xlsx', 'tsv', 'h5ad', 'csv', 'mtx', 'loom', 'soft.gz'} or pass the parameter ext.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Monday, December 4, 2023 at 11:15 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes, the error is because your filenames don't follow the usual convention. Can you rename your files to matrix.mtx.gz and features.tsv.gz and barcodes.tsv.gz ? Or just create symlinks with this name. Then run it again.

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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maximilianh commented 9 months ago

OK, I installed the most current versions of everything, what I found in conda and pip: Python 3.9.5 (conda)

All others with pip --upgrade, so some more recent than yours:

Scipy 1.11.4 scanpy 1.9.6 numpy 1.26.2

and cbScanpy is working just fine on the pbmc3k dataset.

Your error message was this:

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6643, in cbScanpyCli adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname, skipMarkers) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6432, in cbScanpy adata = runSafeRankGenesGroups(adata, clusterField, minCells=5) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4651, in runSafeRankGenesGroups clusterCellCounts = list(adata.obs.groupby([clusterField]).apply(len).iteritems()) File "/usr/local/lib64/python3.9/site-packages/pandas/core/generic.py", line 6204, in getattr return object.getattribute(self, name) AttributeError: 'Series' object has no attribute 'iteritems'

This looks like an installation problem with your pandas package. You can try "pip install pandas --upgrade".

On Mon, Dec 11, 2023 at 10:08 PM bugacov @.***> wrote:

Hi….I was wondering if you were able to reproduce my errors? I think that the main difference seems to be the Python version on my machine but I kinda constrained to stay on that version if possible. Let me know if there’s something else I can try.

Again the versions in my system are:

Python 3.9.18

scanpy==1.9.6

scipy==1.11.3

numpy==1.25.2

Thanks! Alejandro.

From: Alejandro Bugacov @.> Date: Tuesday, December 5, 2023 at 2:34 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Tuesday, December 5, 2023 at 7:23 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Ah, it's just that file. Yes, we do test and it works for me.

This looks a little like a problem with your scanpy installation... but we'll see.

I'm using Python 3.7 and Scanpy 1.9.2 and scipy 1.7.3

I can try to use the same versions that you have. Can you send me the list of these versions: Python, Scipy, Numpy and especially Scanpy.

Python 3.9.18

scanpy==1.9.6

scipy==1.11.3

numpy==1.25.2

Thanks! Alejandro.

On Tue, Dec 5, 2023 at 4:11 PM bugacov @.***> wrote:

wget

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=vfqy0VZ_lZZYeWdNabrBSDEqpde9StRJR_FA1l6CILg&e=>

<

https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e=%3E

-O data/pbmc3k_filtered_gene_bc_matrices.tar.gz<

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=R827nUTL2RtYc_UutyP-51nWeHNYrD9Y0d0nnCisuhc&e=>

<

https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=%3E

Sent from my iPhone

On Dec 5, 2023, at 1:15 AM, Maximilian Haeussler @.***> wrote:

Great, so this almost worked, it's only stuck at the markers calculation step at the end now. This should be easy to fix.

You're using Python 3.9 from 2020 and combine that with the most recent scanpy version, just a few weeks old. We didn't test this combination. You could downgrade scanpy to a few versions older. But yes, it should normally work, it's possible I have to add yet another workaround (I've kept adding workarounds for the different scanpy versions, every few months, something is breaking there).

As soon as I can reproduce this error, I can fix it. Can you tell me where you downloaded the mtx file from? Is this your own file or a 10X pbmc demo file?

On Tue, Dec 5, 2023 at 5:33 AM bugacov @.***> wrote:

I actually uninstall cellbrowser and installed it again and now is doing a bit more but still throwing errors:

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data] cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Loading Scanpy libraries INFO:root:Creating scanpyOut INFO:root:Loading Scanpy libraries INFO:root:cbScanpy $Id$ INFO:root:Command: /usr/local/bin/cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k INFO:root:Matrix input file: filtered_gene_bc_matrices/hg19/matrix.mtx INFO:root:Restricting OPENBLAS to 4 threads INFO:root:Start time: 2023-12-05 04:30:26.199478

anndata 0.10.3 scanpy 1.9.6

PIL 10.0.0 asciitree NA cellbrowser v1.2.3 cffi 1.14.5 colorama 0.4.4 cycler 0.12.1 cython_runtime NA dateutil 2.8.2 entrypoints 0.4 exceptiongroup 1.2.0 fasteners 0.18 get_annotations NA h5py 3.10.0 igraph 0.10.8 importlib_resources NA joblib 1.3.2 kiwisolver 1.4.5 llvmlite 0.41.1 louvain 0.8.1 matplotlib 3.8.2 mpl_toolkits NA natsort 8.4.0 numba 0.58.1 numcodecs 0.11.0 numpy 1.25.2 packaging 23.1 pandas 2.1.3 pkg_resources NA psutil 5.8.0 pyparsing 3.1.1 pytz 2021.1 scipy 1.11.3 session_info 1.0.0 setuptools_scm NA six 1.15.0 sklearn 1.3.2 texttable 1.7.0 threadpoolctl 3.2.0 typing_extensions NA yaml 5.4.1 zarr 2.16.1 zipp NA zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)] Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-05 04:30 INFO:root:Loading Scanpy libraries INFO:root:Loading expression matrix: mtx format INFO:root:Data has 2700 samples/observations INFO:root:Data has 32738 genes/variables INFO:root:Basic filtering: keep only cells with min 200 genes INFO:root:Basic filtering: keep only gene with min 3 cells INFO:root:After filtering: Data has 2700 samples/observations and 13714 genes/variables INFO:root:Making gene names unique INFO:root:'geneIdType' is not specified in config file or set to 'auto'. INFO:root:Auto-detected gene IDs type: symbols INFO:root:Remove cells with more than 0.050000 percent of mitochondrial genes INFO:root:Computing percentage of mitochondrial genes WARNING: saving figure to file scanpyOut/figs/violin.png INFO:root:Remove cells with less than 10 and more than 15000 genes INFO:root:Keeping only cells with < 15000 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Keeping only cells with > 10 genes INFO:root:After filtering: Data has 2643 samples/observations and 13714 genes/variables INFO:root:Expression normalization, counts per cell = 10000 INFO:root:Did log2'ing of data INFO:root:Finding highly variable genes WARNING: saving figure to file scanpyOut/figs/filter_genes_dispersion.png INFO:root:After high-var filtering: Data has 2643 samples/observations and 1844 genes/variables INFO:root:Regressing out percent_mito and number of UMIs INFO:root:Scaling data, max_value=10 INFO:root:Performing initial PCA, number of PCs: 100 WARNING: saving figure to file scanpyOut/figs/pca_variance_ratio.png INFO:root:Estimating number of useful PCs based on Shekar et al, Cell 2016 INFO:root:PC weight cutoff used is (sqrt(# of Genes/# of cells) + 1)^2 INFO:root:See http://www.cell.com/cell/fulltext/S0092-8674(16)31007-8 << https://urldefense.us/v2/url?u=http-3A__www.cell.com_cell_fulltext_S0092-2D8674-2816-2931007-2D8-253C&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=V7ZgmTdvAWsVTOnGdosk-ZKl0Rd7rUdm2Pt_Q236vTI&e=>

https://urldefense.us/v2/url?u=http-3A__www.cell.com_cell_fulltext_S0092-2D8674-2816-2931007-2D8&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=LRCerhXenZ3f2SWEPhr8kULBEGklU2NYVJ-O5nUQ-tA&e=%3E,

STAR methods INFO:root:7 PCs will be used for tSNE and clustering INFO:root:Performing tSNE INFO:root:Running knn, using 7 PCs and 6 neighbors INFO:root:Performing Louvain Clustering, resolution 1.000000 INFO:root:Found 13 louvain clusters INFO:root:Using cluster annotation from field: louvain WARNING: saving figure to file scanpyOut/figs/tsne.png INFO:root:Performing UMAP INFO:root:Performing Layout 'drl' = DrL Distributed Recursive Layout INFO:root:Finding top markers for each cluster INFO:root:Loading Scanpy libraries Traceback (most recent call last): File "/usr/local/bin/cbScanpy", line 8, in sys.exit(cbScanpyCli()) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6643, in cbScanpyCli adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname, skipMarkers) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 6432, in cbScanpy adata = runSafeRankGenesGroups(adata, clusterField, minCells=5) File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4651, in runSafeRankGenesGroups clusterCellCounts = list(adata.obs.groupby([clusterField]).apply(len).iteritems()) File "/usr/local/lib64/python3.9/site-packages/pandas/core/generic.py", line 6204, in getattr return object.getattribute(self, name) AttributeError: 'Series' object has no attribute 'iteritems' facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData/data]

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 7:49 PM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hi….I tried to run it on the example files from Scanpy example given here https://scanpy-tutorials.readthedocs.io/en/latest/pbmc3k.html<< https://urldefense.us/v2/url?u=https-3A__scanpy-2Dtutorials.readthedocs.io_en_latest_pbmc3k.html-253C&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=hkV8fjmu18Vu9JP99fI2Yr2oNegSCXgjEAuaMlSN-ps&e=>

https://urldefense.us/v2/url?u=https-3A__scanpy-2Dtutorials.readthedocs.io_en_latest_pbmc3k.html&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=fwTJ3jAfqNQubzg5eA4igcNVVQaoKw84DwvDYjI16tM&e=%3E

wget

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=vfqy0VZ_lZZYeWdNabrBSDEqpde9StRJR_FA1l6CILg&e=>

<

https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=sVd8NBPPExycR3HnZDVwe3_RsI7oJvmwWVUbzRPIScc&e=%3E

-O data/pbmc3k_filtered_gene_bc_matrices.tar.gz<

http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz%20-O%20data/pbmc3k_filtered_gene_bc_matrices.tar.gz < https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Eqtc9jHwHZU0WqB7tfCK_xU-wHip2pNbZHBHzeETtRHMmpV-ntmbtudYUj4UyfoK&s=R827nUTL2RtYc_UutyP-51nWeHNYrD9Y0d0nnCisuhc&e=>

<

https://urldefense.us/v2/url?u=http-3A__cf.10xgenomics.com_samples_cell-2Dexp_1.1.0_pbmc3k_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-2520-2DO-2520data_pbmc3k-5Ffiltered-5Fgene-5Fbc-5Fmatrices.tar.gz-253E&d=DwQFaQ&c=qzHnJIRvjI6L-clJH8JwLQvf_Iq43fzikf6aoxZgMb8&r=daN-itZmdDhtlnSO-ZKNiA&m=Qi2zRSMbkGew5N7ku4dNpPvlREN86rpFRZ7xEra3aLvTfppUj3r-CiybjUNGKzoV&s=hcBjGM9GQUNRZAiQBn_Kz8qAbb7sSHh-aBH5Suc5hMg&e=%3E

and I cannot get cbScanpy to run on them.

I built the .h5ad file in python using

import numpy as np import pandas as pd import scanpy as sc

adata = sc.read_10x_mtx( 'data/filtered_gene_bc_matrices/hg19/', # the directory with the .mtx file var_names='gene_symbols', # use gene symbols for the variable names (variables-axis index) cache=True) # write a cache file for faster subsequent reading

adata

and then tried to run the

cbImportScanpy -i filtered_gene_bc_matrices-hg19-matrix.h5ad -o pbmc3kImportScanpy

But that give errors too.

I

From: Alejandro Bugacov @.> Date: Monday, December 4, 2023 at 11:36 AM To: maximilianh/cellBrowser @.>, maximilianh/cellBrowser @.> Cc: Author @.> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

I’m not sure if I follow. Your example online points to a file

cbScanpy -e filtered_gene_bc_matrices/hg19/matrix.mtx -o scanpyOut -n pbmc3k

But what you mean here is that I need to put all 3 files under a folder and use that name convention? So I put all 3 files in a folder

facebase-pipeline::root[/var/www/scratch/cellbrowser/cellData] ls -l test1/ total 106608 -rw-r--r--. 1 root root 46082 Dec 4 19:17 barcodes.tsv.gz -rw-r--r--. 1 root root 251118 Dec 4 19:17 features.tsv.gz -rw-r--r--. 1 root root 108861485 Dec 4 18:51 matrix.mtx.gz

And then tried passing the folder name but I get error like below seems like is looking for the h5ad file?

cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e test1 -o scanpyOut -n testOut

INFO:root:Matrix input file: test1

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 19:31:04.112141

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 19:31

INFO:root:Loading Scanpy libraries

INFO:root:Loading expression matrix: mtx format

Traceback (most recent call last):

File "/usr/local/bin/cbScanpy", line 8, in

sys.exit(cbScanpyCli())

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5865, in cbScanpyCli

adata, params = cbScanpy(matrixFname, metaFname, inCluster, confFname, figDir, logFname)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 5407, in cbScanpy

adata = readMatrixAnndata(matrixFname, samplesOnRows=options.samplesOnRows, genome=options.genome)

File "/usr/local/lib/python3.9/site-packages/cellbrowser/cellbrowser.py", line 4803, in readMatrixAnndata

adata = sc.read(matrixFname, cache=False).T

File "/usr/local/lib/python3.9/site-packages/scanpy/readwrite.py", line 127, in read

raise ValueError(

ValueError: Reading with filekey 'test1' failed, the inferred filename PosixPath('write/test1.h5ad') does not exist. If you intended to provide a filename, either use a filename ending on one of the available extensions {'h5', 'anndata', 'txt', 'data', 'mtx.gz', 'tab', 'xlsx', 'tsv', 'h5ad', 'csv', 'mtx', 'loom', 'soft.gz'} or pass the parameter ext.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Monday, December 4, 2023 at 11:15 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes, the error is because your filenames don't follow the usual convention. Can you rename your files to matrix.mtx.gz and features.tsv.gz and barcodes.tsv.gz ? Or just create symlinks with this name. Then run it again.

I am not sure how/if I can solve this, this is a problem in scanpy, as it only accepts the directory name, I think.

On Mon, Dec 4, 2023 at 7:58 PM bugacov @.***> wrote:

Hi….with the 2 mtx.gz files that I tried I did

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

and get that need to specify a dir and not the mtx file for cellranger3-style .mtx

I tried passing the name of a folder w/ the files but didn’t work either.

Can these mtx files be ran by with scanpy?

Thanks,

Alejandro.

cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Loading Scanpy libraries

INFO:root:Loading Scanpy libraries

INFO:root:cbScanpy $Id$

INFO:root:Command: /usr/local/bin/cbScanpy -e bates_matrix.mtx.gz -o scanpyOut -n test1

INFO:root:Matrix input file: bates_matrix.mtx.gz

INFO:root:Restricting OPENBLAS to 4 threads

INFO:root:Start time: 2023-12-04 18:52:21.201824

anndata 0.10.3

scanpy 1.9.6

PIL 10.0.0

asciitree NA

cellbrowser v1.0.1

cffi 1.14.5

colorama 0.4.4

cycler 0.12.1

cython_runtime NA

dateutil 2.8.2

entrypoints 0.4

exceptiongroup 1.2.0

fasteners 0.18

get_annotations NA

h5py 3.10.0

igraph 0.10.8

importlib_resources NA

joblib 1.3.2

kiwisolver 1.4.5

llvmlite 0.41.1

louvain 0.8.1

matplotlib 3.8.2

mpl_toolkits NA

natsort 8.4.0

numba 0.58.1

numcodecs 0.11.0

numpy 1.25.2

packaging 23.1

pandas 2.1.3

pkg_resources NA

psutil 5.8.0

pyparsing 3.1.1

pytz 2021.1

scipy 1.11.3

session_info 1.0.0

setuptools_scm NA

six 1.15.0

sklearn 1.3.2

texttable 1.7.0

threadpoolctl 3.2.0

typing_extensions NA

yaml 5.4.1

zarr 2.16.1

zipp NA

zoneinfo NA

Python 3.9.18 (main, Sep 7 2023, 00:00:00) [GCC 11.4.1 20230605 (Red Hat 11.4.1-2)]

Linux-5.14.0-362.8.1.el9_3.x86_64-x86_64-with-glibc2.34

Session information updated at 2023-12-04 18:52

INFO:root:Loading Scanpy libraries

ERROR:root:For cellranger3-style .mtx files, please specify the directory, not the .mtx.gz file name

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 12:03 PM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Yes try cbScanpy ! It does exactly that !

On Fri, Dec 1, 2023 at 19:01 bugacov @.***> wrote:

Hi…Thanks again for your response. I’m trying to setup a somehow generic process where I take a set of matrix.mtx, features.tsv and barcodes.tsv files and visualize them w/ the cellbrowser. I’m not sure if that contains the UMAP coords and clustering or of they always will so that’s why I thought I had to go thru’ Seurat but if not needed and you can point me to an example on how to go directly to the cellbrowser I’d really appreciate.

In the meantime I’ll try to see if I can get the cbScanpy to work w/ these files! Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Friday, December 1, 2023 at 6:53 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Wait... why are you running Seurat? you're running cbSeurat, Sorry I didn't see that, I was convinced that you're using cbImportSeurat.

I haven't tested cbSeurat in a long time! I should probably retire it.

Don't you have umap coordinates etc as tsv files? If so, you can go directly to cbBuild.

If you need UMAP coords and clustering and cluster markers, and these things are not already there in the meta or separate tsv files, you can also use cbScanpy, which at this point has had a ton more testing.

On Thu, Nov 30, 2023 at 10:35 PM bugacov @.***> wrote:

Hi….The problem is that I’m having a lot of errors when trying to install Seurat 3 or 4. I was able to do it before but seem like it’s failing now. Question: Is there a way of going from the matrix.mtx, features.tsv and barcodes.tsv directly into the cellbrowser w/o converting to Seurat?

Thanks! Alejandro.

From: Maximilian Haeussler @.> Reply-To: maximilianh/cellBrowser @.> Date: Thursday, November 30, 2023 at 11:59 AM To: maximilianh/cellBrowser @.> Cc: Alejandro Bugacov @.>, Author @.***> Subject: Re: [maximilianh/cellBrowser] Issue w/ running cbSeurat: argument "counts" is missing, with no default (Issue #265)

Hm yes I don’t think I’ve tested on Seurat. Do you want share your matrix ? Probably I’ll need to install Seurat5. Can you try it on Seurat 3 or 4 ?

On Thu, Nov 30, 2023 at 19:37 bugacov @.***> wrote:

Hi, I’ve been building my cellbrowsers for matrix files for a while now and was working fine but after some update the pipeline doesn’t seem to be working any more. I’m trying to go from the matrix.mtx.gz barcodes.tsv.gz and features.tsv.gz to the cellbrowser by first building the exprMatrix.tsv.gz using “cbTool mtx2tsv …” and that step seem to work but then next step cbSeurat is failing. Any idea what can be the problem? Is it a problem of using Seurat 5.0.0 w/ latest version of cellbrowser? Thanks! Alejandro.

cbSeurat --exprMatrix=exprMatrix.tsv.gz --outDir=/var/www/scratch/cellbrowser/seurat_temp --name=1W-YOON INFO:root:Creating /var/www/scratch/cellbrowser/seurat_temp INFO:root:Wrote R script to /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R INFO:root:running /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R through Rscript Seurat loaded, version 5.0.0 [1] "Seurat: Reading data" [1] "Seurat: Reading data" [1] "Loading input data matrix" [1] "Loading tsv file with read.delim" [1] "Seurat: Setup" Error in CreateSeuratObject(raw.data = mat, min.cells = 3, min.genes

200) : argument "counts" is missing, with no default Execution halted

real 1m33.191s user 1m17.847s sys 0m1.896s INFO:root:Wrote logfile of R run to /var/www/scratch/cellbrowser/seurat_temp/analysisLog.txt ERROR:root:R script did not complete successfully. Check /var/www/scratch/cellbrowser/seurat_temp/runSeurat.R and analysisLog.txt.

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