Closed akyocum closed 1 year ago
@akyocum So the issue is that you have multiple lines of entries in the same plex,channel combination or sample_name like I could see multiple 30_UT_BP_18A in your file. Is that what directly output from FragPipe? I assume the number of entries in the automatically generated template should be the same as the number of samples in TMT-I report.
It’s due to a fractionation experiment. Each plex was fractionated 5-times before injection into MS.
Anastasia
From: hsiao yi @.> Date: Thursday, February 9, 2023 at 12:51 PM To: Nesvilab/FragPipe-Analyst @.> Cc: Yocum, Anastasia @.>, Mention @.> Subject: Re: [Nesvilab/FragPipe-Analyst] Run names don't match between TMT-I report and annotation (Issue #8) External Email - Use Caution
@akyocumhttps://github.com/akyocum So the issue is that you have multiple lines of entries in the same plex,channel combination or sample_name like I could see multiple 30_UT_BP_18A in your file. Is that what directly output from FragPipe? I assume the number of entries in the automatically generated template should be the same as the number of samples in TMT-I report.
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@akyocum For TMT-I report, we don't separate the fractionated results. That's why I wonder if FragPipe generated that or you created it by yourself. To make it compatible, match the number of sample_name in your experimental annotation as the number of samples available in the TMT-I report.
Sorry, I didn't answer your question directly.
Yes, this annotation file was automatically generated from FragPipe, I edited that provided annotation.txt file to have the header / columns needed per the documentation.
I too would assume that the number of entries in the template should match in the TMT-I report, but it's not the case.
In each plex directory, the psm.txt file has all files:scanNumbers representing all fractions in that specific plex. The column headers in these individual plex psm file have coalesced the fractions into one sample name. Makes sense.
Then, the combined_psm.txt file also lists as rows the scan numbers and all samples across all plexes as column headers. There are appropriately 'zeros' in the observations when that file:scanNumber (plex/fraction) is not in that sample.
Then, the TMT-I report coalesced to just have column headers to have all samples in all plexes. But the annotation file has all fractions in each plex listed out iteratively.
Maybe the path forward is to list one of the fractions and one label?
Anastasia
Oppps.. we posted at the same time. But same conclusion.
So, for channel / plex just slot in a number?
Anastasia
@akyocum could you post the manifest file you have for your FragPipe run? We kind of don't know why that would happen. For channel, that will be 126, 127N etc. For plex, you could put whatever you want, like I usually have TMT_01 for first plex-set.
Certainly,
Removing the fraction rows so that there was one row per sample name worked however.
I feel this wasn't an optimized set up.
Experimental design is complex however, five Plex, with 5-fractions each, TMT-16 with two pooled controls. There are biological replicates, but needed to keep each individual file separate for downstream analysis.
@akyocum May I asked which FragPipe version you are using? I don't think your experimental design is too complex. In CPTAC projects, we have similar setting that multiple fractions are collected for each samples.
Yep,
FragPipe v 19.1
Hi @akyocum ,
Can you also upload the log file with the name starts with log-
?
Thanks,
Fengchao
There are duplicated "samples" in the annotation.txt files, which results in "duplicated" rows in the experiment_annotation.txt
file.
~~~~~~annotation files~~~~~~~
E:\Oshea_UPMC_TreatedOrganoids_phospho\mzml4FragPipe\plex1\Plex1_annotation.txt:
Channel annotation
126 60_CHIR_BP_11E
127C 60_DMSO_BP_17A
127N 60_CHIR_BP_17A
128C 60_IWR_BP_18A
128N 60_IWR_BP_19D
129C 90_UT_BP_11E
129N 90_UT_BP_17A
130C 90_DMSO_C_9G
130N 90_CHIR_C_9G
131C 90_IWR_BP_11E
131N 30_UT_BP_18A
132C 60_UT_C_10V
132N 60_UT_BP_11E
133N 60_DMSO_C_10V
133C pool_16
134N bridge_16
126 60_CHIR_BP_11E
127C 60_DMSO_BP_17A
127N 60_CHIR_BP_17A
128C 60_IWR_BP_18A
128N 60_IWR_BP_19D
129C 90_UT_BP_11E
129N 90_UT_BP_17A
130C 90_DMSO_C_9G
130N 90_CHIR_C_9G
131C 90_IWR_BP_11E
131N 30_UT_BP_18A
132C 60_UT_C_10V
132N 60_UT_BP_11E
133N 60_DMSO_C_10V
133C pool_17
134N bridge_17
126 60_CHIR_BP_11E
127C 60_DMSO_BP_17A
127N 60_CHIR_BP_17A
128C 60_IWR_BP_18A
128N 60_IWR_BP_19D
129C 90_UT_BP_11E
129N 90_UT_BP_17A
130C 90_DMSO_C_9G
130N 90_CHIR_C_9G
131C 90_IWR_BP_11E
131N 30_UT_BP_18A
132C 60_UT_C_10V
132N 60_UT_BP_11E
133N 60_DMSO_C_10V
133C pool_18
134N bridge_18
126 60_CHIR_BP_11E
127C 60_DMSO_BP_17A
127N 60_CHIR_BP_17A
128C 60_IWR_BP_18A
128N 60_IWR_BP_19D
129C 90_UT_BP_11E
129N 90_UT_BP_17A
130C 90_DMSO_C_9G
130N 90_CHIR_C_9G
131C 90_IWR_BP_11E
131N 30_UT_BP_18A
132C 60_UT_C_10V
132N 60_UT_BP_11E
133N 60_DMSO_C_10V
133C pool_19
134N bridge_19
126 60_CHIR_BP_11E
127C 60_DMSO_BP_17A
127N 60_CHIR_BP_17A
128C 60_IWR_BP_18A
128N 60_IWR_BP_19D
129C 90_UT_BP_11E
129N 90_UT_BP_17A
130C 90_DMSO_C_9G
130N 90_CHIR_C_9G
131C 90_IWR_BP_11E
131N 30_UT_BP_18A
132C 60_UT_C_10V
132N 60_UT_BP_11E
133N 60_DMSO_C_10V
133C pool_20
134N bridge_20
E:\Oshea_UPMC_TreatedOrganoids_phospho\mzml4FragPipe\plex2\Plex2_annotation.txt:
Channel annotation
126 90_CHIR_BP_11E
127C 90_IWR_BP_17A
127N 90_IWR_BP_18A
128C 90_UT_BP_18A
128N 90_IWR_C_8WW
129C 90_DMSO_BP_11E
129N 60_UT_BP_17A
130C 60_UT_BP_18A
130N 60_DMSO_C_5G
131C 60_IWR_C_5G
131N 60_IWR_C_8WW
132C 60_CHIR_BP_18A
132N 60_DMSO_BP_19D
133N 60_CHIR_BP_19D
133C pool_6
134N bridge_6
126 90_CHIR_BP_11E
127C 90_IWR_BP_17A
127N 90_IWR_BP_18A
128C 90_UT_BP_18A
128N 90_IWR_C_8WW
129C 90_DMSO_BP_11E
129N 60_UT_BP_17A
130C 60_UT_BP_18A
130N 60_DMSO_C_5G
131C 60_IWR_C_5G
131N 60_IWR_C_8WW
132C 60_CHIR_BP_18A
132N 60_DMSO_BP_19D
133N 60_CHIR_BP_19D
133C pool_7
134N bridge_7
126 90_CHIR_BP_11E
127C 90_IWR_BP_17A
127N 90_IWR_BP_18A
128C 90_UT_BP_18A
128N 90_IWR_C_8WW
129C 90_DMSO_BP_11E
129N 60_UT_BP_17A
130C 60_UT_BP_18A
130N 60_DMSO_C_5G
131C 60_IWR_C_5G
131N 60_IWR_C_8WW
132C 60_CHIR_BP_18A
132N 60_DMSO_BP_19D
133N 60_CHIR_BP_19D
133C pool_8
134N bridge_8
126 90_CHIR_BP_11E
127C 90_IWR_BP_17A
127N 90_IWR_BP_18A
128C 90_UT_BP_18A
128N 90_IWR_C_8WW
129C 90_DMSO_BP_11E
129N 60_UT_BP_17A
130C 60_UT_BP_18A
130N 60_DMSO_C_5G
131C 60_IWR_C_5G
131N 60_IWR_C_8WW
132C 60_CHIR_BP_18A
132N 60_DMSO_BP_19D
133N 60_CHIR_BP_19D
133C pool_9
134N bridge_9
126 90_CHIR_BP_11E
127C 90_IWR_BP_17A
127N 90_IWR_BP_18A
128C 90_UT_BP_18A
128N 90_IWR_C_8WW
129C 90_DMSO_BP_11E
129N 60_UT_BP_17A
130C 60_UT_BP_18A
130N 60_DMSO_C_5G
131C 60_IWR_C_5G
131N 60_IWR_C_8WW
132C 60_CHIR_BP_18A
132N 60_DMSO_BP_19D
133N 60_CHIR_BP_19D
133C pool_10
134N bridge_10
E:\Oshea_UPMC_TreatedOrganoids_phospho\mzml4FragPipe\plex3\Plex3_annotation.txt:
Channel annotation
126 30_UT_C_5G
127C 60_UT_BP_19D
127N 60_CHIR_C_5G
128C 60_DMSO_C_8WW
128N 60_CHIR_C_8WW
129C 60_IWR_C_9G
129N 60_DMSO_BP_11E
130C 90_UT_BP_19D
130N 90_DMSO_C_8WW
131C 90_IWR_C_10V
131N 90_CHIR_BP_17A
132C 90_DMSO_BP_18A
132N 90_CHIR_BP_18A
133N 90_IWR_BP_19D
133C pool_1
134N bridge_1
126 30_UT_C_5G
127C 60_UT_BP_19D
127N 60_CHIR_C_5G
128C 60_DMSO_C_8WW
128N 60_CHIR_C_8WW
129C 60_IWR_C_9G
129N 60_DMSO_BP_11E
130C 90_UT_BP_19D
130N 90_DMSO_C_8WW
131C 90_IWR_C_10V
131N 90_CHIR_BP_17A
132C 90_DMSO_BP_18A
132N 90_CHIR_BP_18A
133N 90_IWR_BP_19D
133C pool_2
134N bridge_2
126 30_UT_C_5G
127C 60_UT_BP_19D
127N 60_CHIR_C_5G
128C 60_DMSO_C_8WW
128N 60_CHIR_C_8WW
129C 60_IWR_C_9G
129N 60_DMSO_BP_11E
130C 90_UT_BP_19D
130N 90_DMSO_C_8WW
131C 90_IWR_C_10V
131N 90_CHIR_BP_17A
132C 90_DMSO_BP_18A
132N 90_CHIR_BP_18A
133N 90_IWR_BP_19D
133C pool_3
134N bridge_3
126 30_UT_C_5G
127C 60_UT_BP_19D
127N 60_CHIR_C_5G
128C 60_DMSO_C_8WW
128N 60_CHIR_C_8WW
129C 60_IWR_C_9G
129N 60_DMSO_BP_11E
130C 90_UT_BP_19D
130N 90_DMSO_C_8WW
131C 90_IWR_C_10V
131N 90_CHIR_BP_17A
132C 90_DMSO_BP_18A
132N 90_CHIR_BP_18A
133N 90_IWR_BP_19D
133C pool_4
134N bridge_4
126 30_UT_C_5G
127C 60_UT_BP_19D
127N 60_CHIR_C_5G
128C 60_DMSO_C_8WW
128N 60_CHIR_C_8WW
129C 60_IWR_C_9G
129N 60_DMSO_BP_11E
130C 90_UT_BP_19D
130N 90_DMSO_C_8WW
131C 90_IWR_C_10V
131N 90_CHIR_BP_17A
132C 90_DMSO_BP_18A
132N 90_CHIR_BP_18A
133N 90_IWR_BP_19D
133C pool_5
134N bridge_5
E:\Oshea_UPMC_TreatedOrganoids_phospho\mzml4FragPipe\plex4\Plex4_annotation.txt:
Channel annotation
126 90_DMSO_C_5G
127C 90_IWR_C_5G
127N 90_CHIR_BP_19D
128C 60_UT_C_5G
128N 60_CHIR_C_9G
129C 60_IWR_C_10V
129N 30_UT_C_9G
130C 30_UT_C_10V
130N 60_DMSO_BP_18A
131C 90_UT_C_5G
131N 90_UT_C_8WW
132C 90_DMSO_C_10V
132N 90_CHIR_C_10V
133N 90_DMSO_BP_17A
133C pool_21
134N bridge_21
126 90_DMSO_C_5G
127C 90_IWR_C_5G
127N 90_CHIR_BP_19D
128C 60_UT_C_5G
128N 60_CHIR_C_9G
129C 60_IWR_C_10V
129N 30_UT_C_9G
130C 30_UT_C_10V
130N 60_DMSO_BP_18A
131C 90_UT_C_5G
131N 90_UT_C_8WW
132C 90_DMSO_C_10V
132N 90_CHIR_C_10V
133N 90_DMSO_BP_17A
133C pool_22
134N bridge_22
126 90_DMSO_C_5G
127C 90_IWR_C_5G
127N 90_CHIR_BP_19D
128C 60_UT_C_5G
128N 60_CHIR_C_9G
129C 60_IWR_C_10V
129N 30_UT_C_9G
130C 30_UT_C_10V
130N 60_DMSO_BP_18A
131C 90_UT_C_5G
131N 90_UT_C_8WW
132C 90_DMSO_C_10V
132N 90_CHIR_C_10V
133N 90_DMSO_BP_17A
133C pool_23
134N bridge_23
126 90_DMSO_C_5G
127C 90_IWR_C_5G
127N 90_CHIR_BP_19D
128C 60_UT_C_5G
128N 60_CHIR_C_9G
129C 60_IWR_C_10V
129N 30_UT_C_9G
130C 30_UT_C_10V
130N 60_DMSO_BP_18A
131C 90_UT_C_5G
131N 90_UT_C_8WW
132C 90_DMSO_C_10V
132N 90_CHIR_C_10V
133N 90_DMSO_BP_17A
133C pool_24
134N bridge_24
126 90_DMSO_C_5G
127C 90_IWR_C_5G
127N 90_CHIR_BP_19D
128C 60_UT_C_5G
128N 60_CHIR_C_9G
129C 60_IWR_C_10V
129N 30_UT_C_9G
130C 30_UT_C_10V
130N 60_DMSO_BP_18A
131C 90_UT_C_5G
131N 90_UT_C_8WW
132C 90_DMSO_C_10V
132N 90_CHIR_C_10V
133N 90_DMSO_BP_17A
133C pool_25
134N bridge_25
E:\Oshea_UPMC_TreatedOrganoids_phospho\mzml4FragPipe\plex5\Plex5_annotation.txt:
Channel annotation
126 90_CHIR_C_8WW
127C 90_IWR_C_9G
127N 90_DMSO_BP_19D
128C 30_UT_BP_11E
128N 30_UT_BP_17A
129C 60_UT_C_8WW
129N 60_UT_C_9G
130C 60_DMSO_C_9G
130N 60_CHIR_C_10V
131C 60_IWR_BP_11E
131N 60_IWR_BP_17A
132C 90_UT_C_9G
132N 90_UT_C_10V
133N 90_CHIR_C_5G
133C pool_11
134N bridge_11
126 90_CHIR_C_8WW
127C 90_IWR_C_9G
127N 90_DMSO_BP_19D
128C 30_UT_BP_11E
128N 30_UT_BP_17A
129C 60_UT_C_8WW
129N 60_UT_C_9G
130C 60_DMSO_C_9G
130N 60_CHIR_C_10V
131C 60_IWR_BP_11E
131N 60_IWR_BP_17A
132C 90_UT_C_9G
132N 90_UT_C_10V
133N 90_CHIR_C_5G
133C pool_12
134N bridge_12
126 90_CHIR_C_8WW
127C 90_IWR_C_9G
127N 90_DMSO_BP_19D
128C 30_UT_BP_11E
128N 30_UT_BP_17A
129C 60_UT_C_8WW
129N 60_UT_C_9G
130C 60_DMSO_C_9G
130N 60_CHIR_C_10V
131C 60_IWR_BP_11E
131N 60_IWR_BP_17A
132C 90_UT_C_9G
132N 90_UT_C_10V
133N 90_CHIR_C_5G
133C pool_13
134N bridge_13
126 90_CHIR_C_8WW
127C 90_IWR_C_9G
127N 90_DMSO_BP_19D
128C 30_UT_BP_11E
128N 30_UT_BP_17A
129C 60_UT_C_8WW
129N 60_UT_C_9G
130C 60_DMSO_C_9G
130N 60_CHIR_C_10V
131C 60_IWR_BP_11E
131N 60_IWR_BP_17A
132C 90_UT_C_9G
132N 90_UT_C_10V
133N 90_CHIR_C_5G
133C pool_14
134N bridge_14
126 90_CHIR_C_8WW
127C 90_IWR_C_9G
127N 90_DMSO_BP_19D
128C 30_UT_BP_11E
128N 30_UT_BP_17A
129C 60_UT_C_8WW
129N 60_UT_C_9G
130C 60_DMSO_C_9G
130N 60_CHIR_C_10V
131C 60_IWR_BP_11E
131N 60_IWR_BP_17A
132C 90_UT_C_9G
132N 90_UT_C_10V
133N 90_CHIR_C_5G
133C pool_15
134N bridege_15
~~~~~~~~~~~~~~~~~~~~~~
Best,
Fengchao
Ok, I think this makes more sense to me. I was creating annotations like MSstats requires where each fraction in each plex would be listed. I've removed all the duplicates and rerunning now.
@akyocum I will close this now. Please let us know if you still meet some issues. Thanks.
Hey there,
Now working through a multi-plex, multi-fraction, TMT experiment. The error I receive is this:
I've reviewed the documentation that shows how to manipulate the annotation file that is outputted from FragPipe and it looks like I'm ok in that regard. I checked and the column headers between the
annotation.tsv
andabundance_gene_MD.tsv
and they do indeed match. I'm attaching the annotation file and the head of the abundance_gene_MD.tsv file for troubleshooting.Thanks for your help!
Anastasia
experiment_annotation.txt abundance_gene_MD_4issue.txt