hyunminkang
commented
5 years ago This is not necessary a concern. Cell Ranger sometimes do not assign cell barcodes when it is not certain.
Hyun.
Hyun Min Kang, Ph.D. Associate Professor of Biostatistics University of Michigan, Ann Arbor Email : hmkang@umich.edu
On Tue, May 28, 2019 at 2:22 AM Anne Senabouth notifications@github.com wrote:
Hello,
We have been noticing the following warning messages during the analysis of data generated by Cell Ranger 3.
The following parameters are available. Ones with "[]" are in effect: Options for input SAM/BAM/CRAM : --sam [/share/ScratchGeneral/annsen/data/experimental_data/CLEAN/POAG_scRNA/POAG_scRNA_V2/181108_MD_003/181108_MD_003/outs/possorted_genome_bam.bam], --tag-group [CB], --tag-UMI [UB] Options for input VCF/BCF : --vcf [/share/ScratchGeneral/annsen/data/experimental_data/CLEAN/POAG_scRNA/POAG_scRNA.vcf], --field [GP], --geno-error [0.01], --min-mac [1], --min-callrate [0.50], --sm, --sm-list [/share/ScratchGeneral/annsen/repositories/POAG_scRNA/Pools/181108_MD_003.tsv] Output Options : --out [/share/ScratchGeneral/annsen/analysis/POAG_scRNA/POAG_scRNA_Demuxlet/181108_MD_003], --alpha [0.00, 0.50], --write-pair, --doublet-prior [0.50], --sam-verbose [1000000], --vcf-verbose [10000] Read filtering Options : --cap-BQ [40], --min-BQ [13], --min-MQ [20], --min-TD, --excl-flag [3844] Cell/droplet filtering options : --group-list [/share/ScratchGeneral/annsen/data/experimental_data/CLEAN/POAG_scRNA/POAG_scRNA_V2/181108_MD_003/181108_MD_003/outs/filtered_feature_bc_matrix/barcodes.tsv], --min-total, --min-uniq, --min-snp
Run with --help for more detailed help messages of each argument.
NOTICE [2019/05/24 17:19:52] - Finished loading 12569 droplet/cell barcodes to consider NOTICE [2019/05/24 17:19:52] - Finished loading 7 IDs from /share/ScratchGeneral/annsen/repositories/POAG_scRNA/Pools/181108_MD_003.tsv NOTICE [2019/05/24 17:19:52] - Finished identifying 7 samples to load from VCF/BCF NOTICE [2019/05/24 17:19:55] - Reading 0 reads at 1:1 and skipping 0 NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 12274-th read A00152:78:HHNG5DSXX:4:2103:4155:7310 at 1:17475-17573. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 26628-th read A00152:78:HHNG5DSXX:4:1669:22733:7764 at 1:128604-128702. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 26651-th read A00152:78:HHNG5DSXX:1:2140:16242:34976 at 1:128667-128765. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 26656-th read A00152:78:HHNG5DSXX:2:1412:32208:27398 at 1:128679-128777. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 26996-th read A00152:78:HHNG5DSXX:2:2268:17779:12868 at 1:134158-134256. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 26997-th read A00152:78:HHNG5DSXX:2:2526:30228:26318 at 1:134158-134256. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 29536-th read A00152:78:HHNG5DSXX:4:2647:3405:22701 at 1:135153-135251. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 29577-th read A00152:78:HHNG5DSXX:4:2253:7473:12117 at 1:138510-138608. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 35070-th read A00152:78:HHNG5DSXX:2:1137:1271:5259 at 1:157470-692645. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Cannot find Droplet/Cell tag CB from 35117-th read A00152:78:HHNG5DSXX:1:2460:9408:17973 at 1:157515-692690. Treating all of them as a single group NOTICE [2019/05/24 17:19:55] - WARNING: Suppressing 10+ missing Droplet/Cell tag warnings...
This is the first time we have seen such issues; we did not encounter them with data generated by Cell Ranger 2. It is of great concern to us as the results have less cells.
This version of demuxlet was installed via anaconda. The conda environment is as follows:
packages in environment at /share/ClusterShare/software/contrib/annsen/anaconda3/envs/demuxlet:
#
Name Version Build Channel
bzip2 1.0.6 h14c3975_1002 conda-forge ca-certificates 2019.3.9 hecc5488_0 conda-forge curl 7.64.0 h646f8bb_0 conda-forge demuxlet 1.0 h7279bd8_1 bioconda htslib 1.9 ha228f0b_7 bioconda krb5 1.16.3 hc83ff2d_1000 conda-forge libcurl 7.64.0 h01ee5af_0 conda-forge libdeflate 1.0 h14c3975_1 bioconda libedit 3.1.20170329 hf8c457e_1001 conda-forge libgcc-ng 8.2.0 hdf63c60_1 anaconda libssh2 1.8.0 h1ad7b7a_1003 conda-forge libstdcxx-ng 8.2.0 hdf63c60_1 anaconda libtool 2.4.6 h14c3975_1002 conda-forge ncurses 6.1 hf484d3e_1002 conda-forge openssl 1.0.2r h14c3975_0 conda-forge samtools 1.9 h43f6869_9 bioconda tk 8.6.9 h84994c4_1001 conda-forge xz 5.2.4 h14c3975_1001 conda-forge zlib 1.2.11 h14c3975_1004 conda-forge
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asenabouth
Hello,
We have been noticing the following warning messages during the analysis of data generated by Cell Ranger 3.
This is the first time we have seen such issues; we did not encounter them with data generated by Cell Ranger 2. It is of great concern to us as the results have less cells.
This version of demuxlet was installed via anaconda. The conda environment is as follows: