bam file missing barcode and UMI info
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3 months ago
malonzm1 • 0

Hi,

I used STARsolo but my bam files, below, are missing barcode and UMI info:

(/scratch/work/malonzm1/.conda_envs/scvi-env) [malonzm1@login3]/scratch/cs/pan-autoimmune/data/star/scRNAseq/GSE151177/SRR11848679% samtools view SRR11848679Aligned.out.bam|head -n5
NB500961:910:HJ5TWBGXC:1:23304:10007:9225       0       chr1    6100902 255     55M     *       0       0       TTCGGAGCCCCCACTGTTTCCCACTCAGCTTTGTGCTCAGATCCCAGGTCCCAAG   AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE NH:i:1  HI:i:1  AS:i:54 nM:i:0  NM:i:0  MD:Z:55 jM:B:c,-1 jI:B:i,-1
NB500961:910:HJ5TWBGXC:1:13111:23716:1741       0       chr1    6100902 255     4S51M   *       0       0       GAGATTCGGAGCCCACACTGTTTCCCACTCAGCTTTGTGCTAATATCCAAGGTCC   ///AA/EEE/AEEE//AE/////E/<A<EE/E/AE///E/E/A/EEE</A<AA// NH:i:1  HI:i:1  AS:i:42 nM:i:4  NM:i:4  MD:Z:10C26C1G4C6  jM:B:c,-1       jI:B:i,-1
NB500961:910:HJ5TWBGXC:1:22211:15746:1375       0       chr1    6100902 255     55M     *       0       0       TTCGGAGCCCCCACTGTTTCCCACTCAGCTTTGTGCTCAGATCCCAGGTCCCAAG   AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEE NH:i:1  HI:i:1  AS:i:54 nM:i:0  NM:i:0  MD:Z:55 jM:B:c,-1 jI:B:i,-1
NB500961:910:HJ5TWBGXC:1:13309:24262:17947      0       chr1    6100903 255     55M     *       0       0       TCGGAGCCCCCACTGTTTCCCACTCAGCTTTGTGCTCAGATCCCAGGTCCCAAGG   AAAAAEAAA/EAEEEEEEE/EEEEEEEEEEEEEEEEAEAEEAEEEEEEEEEEEEE NH:i:1  HI:i:1  AS:i:54 nM:i:0  NM:i:0  MD:Z:55 jM:B:c,-1 jI:B:i,-1

I used the following script:

'STAR --outSAMattributes All --outSAMtype BAM Unsorted --quantMode GeneCounts --readFilesCommand gunzip -c --runThreadN %s --sjdbGTFfile %s --outReadsUnmapped Fastx --outMultimapperOrder Random --genomeDir %s --readFilesIn %s %s --outFileNamePrefix %s --soloType CB_UMI_Simple --soloBarcodeReadLength 0 --soloCBwhitelist %s --soloUMIlen %s --soloCBlen %s --soloUMIstart %s'%(NCPU, GTFFILE, GENOMEDIR, infile2, infile1, OUTPREFIX, whitelist, soloumilen, solocblen, soloumistart)

Please advise.

Thanks and good day.

barcode star starsolo • 584 views
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What is %s pointing to in --soloCBwhitelist %s? It should point to a whitelist file and not a directory assuming that is what you are doing. Are you running this under SLURM?

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Thanks. %s is pointing to a whitelist file. I am running this under SLURM.

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It must be pointing to a directory since you have used it in multiple locations in your command line. It needs to directly point to a file. BTW I have never seen % used in actual program command line. Far as I know % is used only in the SLURM commands.

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I now use "--outSAMattributes NH HI AS nM NM MD jM jI MC ch XS CR UR GX GN sM CB UB" instead of "--outSAM attributes All". Re %s, I actually use python, using os.system.

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So has this fixed the problem?

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