Closed chilampoon closed 1 year ago
Hi, I am not the one that processed that 10x data so I am not a 100% sure about the processing, but I can refer you here: https://github.com/tegowski/scDARTHEKcells for the processing info; and here https://pubmed.ncbi.nlm.nih.gov/36042888/ for the detailed protocol.
As far as the script requirements for 10x data, the bam file should include the "CB:Z:" tag, which is the Chromium cellular barcode sequence (error corrected). I would think this is included with the cell ranger bam. However, I think the bam file used for 10x data was generated with STAR directly and not with cell ranger (see GitHub link above).
If these don't answer your questions, please let me know, I can dig further to see why it's not working. If you would want it to work on the cell ranger bam file and it does not, I can try to modify the script so it does.
Hi @mflamand thanks for the reply! I was actually following that repo and running the perl scripts and got empty matrices. (also seems like only smart-seq2 processings there, no 10x). That's why I wanted to ask here to see if you've made changes in recent updates for the scripts.
One more dump q - did you trim the 10x data as well? I think it's just TSO (?) cz in the paper you said you directly use outputs from cellranger... and cellranger v4.0+ clipped TSO and polyA/T maybe @tegowski know it better? thank you!
Hello,
I can also help try and figure out why you got empty matrices from the 10x data, but not smart-seq. I could help if you send me your script used to call parseBAM.pl and maybe a screenshot of
But as far as the other question, you are correct. I used cellranger 3.1, but it trims TSO and polyA, but after the cellranger alignment with STAR, I did no further trimming prior to using the bamfiles for Bullseye.
Let me know if you have any other questions! Matt
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From: @.> Sent: Monday, November 21, 2022 5:43 PM To: @.> Cc: Matt Tegowski, @.>; @.> Subject: Re: [mflamand/Bullseye] empty output from parseBAM.pl (Issue #8)
Hi @mflamandhttps://urldefense.com/v3/__https:/github.com/mflamand__;!!OToaGQ!vEWWuO9OoV4xDR0yBCvM9GRvrgXDrPhD8g0uKJLc1r7u_3UFTvvbjip0cmjJGr8TGUs9GWxs_dczrOVg_kkXbxvjlkna14G5$ thanks for the reply! I was actually following that repo and running the perl scripts and got empty matrices. (also seems like only smart-seq2 processings there, no 10x). That's why I wanted to ask here to see if you've made changes in recent updates for the scripts.
One more dump q - did you trim the 10x data as well? I think it's just TSO (?) cz in the paper you said you directly use outputs from cellranger... and cellranger v4.0+ clipped TSO and polyA/T maybe @tegowskihttps://urldefense.com/v3/__https:/github.com/tegowski__;!!OToaGQ!vEWWuO9OoV4xDR0yBCvM9GRvrgXDrPhD8g0uKJLc1r7u_3UFTvvbjip0cmjJGr8TGUs9GWxs_dczrOVg_kkXbxvjltiPCxAW$ know it better? thank you!
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Thanks @tegowski - I actually found quite many tso or partial tso in the reads, I am gonna trim tso in R2 fastq and align them using HISAT3N and then try to run your perl scripts again to see what will happen
Did you use Hisat to align previously? And if so, did you check that the barcodes are in the CB:Z: field of the bamfile? That could be one reason its coming out blank is if the barcodes are not there or in a different field Bullseye may have no output.
Matt
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From: @.> Sent: Tuesday, November 22, 2022 1:53 PM To: @.> Cc: Matt Tegowski, @.>; @.> Subject: Re: [mflamand/Bullseye] empty output from parseBAM.pl (Issue #8)
Thanks @tegowskihttps://urldefense.com/v3/__https:/github.com/tegowski__;!!OToaGQ!rvvgfu1h0N6A2xEmADlBcJ4EHxmBp3M1PS5SOplXDshe7sOcq0BchHdXnW9ZxJLOp3fFVz66Dx9ACgS8yV9KYYmUgDRL5GDq$ - I actually found quite many tso or partial tso in the reads, I am gonna trim tso in R2 fastq and align them using HISAT3N and then try to run your perl scripts again to see what will happen
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Did you use Hisat to align previously? And if so, did you check that the barcodes are in the CB:Z: field of the bamfile? That could be one reason its coming out blank is if the barcodes are not there or in a different field Bullseye may have no output. Matt Sent from Mailhttps://go.microsoft.com/fwlink/?LinkId=550986 for Windows From: @.***> Sent: Tuesday,
I tried cellranger bams and minimap2 bams and both got empty matix output.. I think barcode tags are in cellranger bams. But I am aware of adding barcode & umi tags back to the alignment outputs
Oh actually the barcode tag name is CB
not CB:Z
in my cellranger bams, is that an issue?
I think that is likely to be an issue if you are using the “10x” option for the barcode because it is specifically looking for the “CB:Z:” pattern. But there is an option to input your own pattern as REGEX instead of using the 10x or SMART options. You should be able to input “CB:” instead
Matt
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Oh actually the barcode tag name is CB not CB:Z in my cellranger bams, is that an issue?
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Hi,
as Matt said, you can indicate the actual sam tag which correspond to the cell ID instead of the 10x (which is hardcoded for CB:Z:) or SMART (which is hardcoded for RG:Z:).
I could modify the script so it also recognize "CB:" as well if that is the new output of cellranger. If you would prefer this, could you please 10 or so lines of the bam file so I can test it work properly?
Best,
Sure.
I just noticed that it's not CB:
but still CB:Z:
in cellranger bam, however tags after pysam processing it'll be like ('CB', 'AGTGCCGCAAGACGGT-1')
, that's why I was saying the tag name seemed different yesterday.
So maybe there're some other issues that caused empty matrices...
Attaching some of the alignments:
A00814:412:H37KTDSX2:1:2218:29026:29966 16 chr1 3019471 0 75M15S * 0 0 CTGGGCTGGAATTTGTGTTCTCTTAGTGTCTGTATAACATCTGTCCAGGCTCTTCTGGCTTTCATAGTCTCTGCGTTGATACCACTGCTT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF NH:i:6 HI:i:5 AS:i:73 nM:i:0 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:ACCTGAATCTCACCCA CY:Z:FFFFFFFFFFFFFFFF CB:Z:ACCTGAATCTCACCCA-1 UR:Z:ATTCGCTTCCCG UY:Z:FFFFFFFFFFFF UB:Z:ATTCGCTTCCCG
A00814:412:H37KTDSX2:1:1346:11559:36886 0 chr1 3040263 255 4S86M * 0 0 GTTGCAACATTAAGAATGGAAAGCCAACATTCACGTGGAAACTGAACAACACTCTTCTCAATGATACCTTGGTCAAGGAAGGAATAAAGA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFF NH:i:1 HI:i:1 AS:i:78 nM:i:3 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:TCAGTTTTCCATGAGT CY:Z:FFFFFFFFFFFFFFFF CB:Z:TCAGTTTTCCATGAGT-1 UR:Z:AGCCAATTCGAC UY:Z:FFFF:FFFFFFF UB:Z:AGCCAATTCGAC
A00814:412:H37KTDSX2:1:1371:32036:17644 16 chr1 3043192 3 4S86M * 0 0 AGTTATGTTTACAGTATATCAGGGAGATCTACACAATGGAGTACTACTCAGCTATTAAAAAGAATGAATTTATGAAATTCCTAGCCAAAT :,,,,F,,,,,,,F,,,:,,,,,,F,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF NH:i:2 HI:i:1 AS:i:70 nM:i:7 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:CCGGGTATCGCTGATA CY:Z:FFFFFFFFFFFFFFFF CB:Z:CCGGGTATCGCTGATA-1 UR:Z:TTTTGCATGGGT UY:Z:FFFFFFFFFFFF UB:Z:TTTTGCATGGGT
A00814:412:H37KTDSX2:1:1120:10312:8390 0 chr1 3050822 255 90M * 0 0 ATCAGATCCCATATAGATGATTGTGAGCCACCACATGGGTGCTGGGAATTGAACTCAGAATCTCTGAAAGAGCAGCCAGTGTTCTTAACC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:88 nM:i:0 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:ATGGAGGGTGTGTGTG CY:Z:FFFFFFFFFFFFFFFF CB:Z:ATGGAGGGTGTGTTTG-1 UR:Z:TGTGTGTGTGTG UY:Z:FFFFFFFFFFFF UB:Z:TGTGTGTGTGTG
A00814:412:H37KTDSX2:1:2248:7952:18490 16 chr1 3063421 255 87M3S * 0 0 GATCTCATTATTGGTAGTTGTGAGCTACCATGTGGTTGCTTGGTTTTGAACTGAGGACATTTGGATGAGCAGTCGGATGCTCTTTCCGTG FFF:FF,FFF,,FF:FFF,FFF,F,F:F,FFFFFFF:FFF,::,FFFFF,FF,F:FF,,,,FFFF,,FF,,:F,FF,:,FF,,:,F,,F, NH:i:1 HI:i:1 AS:i:69 nM:i:8 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:GGGCTACTCCGACATA CY:Z:FFF::FFFFFFFFFFF CB:Z:GGGCTACTCCGACATA-1 UR:Z:CAGCAGAAATGA UY:Z:FFFFFFFF:FFF UB:Z:CAGCAGAAATGA
A00814:412:H37KTDSX2:2:2638:22598:22467 16 chr1 3063470 255 60M30S * 0 0 ACTCAGGACCTTCGGAAGAGCAGTCGGATGCTCTTACCCACTGAGCCATTTCACCAGCCCCCCATGTACTCTGCGTTGATACCACTGCTT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:59 nM:i:0 ts:i:30 RG:Z:control_2:0:1:H37KTDSX2:2 RE:A:I xf:i:0 CR:Z:TCGCTCAGTGGCTAGA CY:Z:FFFFFFFFFFFFFFFF CB:Z:TCGCTCAGTGGCTAGA-1 UR:Z:GATTTTTGGTTT UY:Z:FFFFFFF:F,FF UB:Z:GATTTTTGGTTT
A00814:412:H37KTDSX2:2:2638:23375:24064 16 chr1 3063470 255 60M30S * 0 0 ACTCAGGACCTTCGGAAGAGCAGTCGGATGCTCTTACCCACTGAGCCATTTCACCAGCCCCCCATGTACTCTGCGTTGATACCACTGCTT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:59 nM:i:0 ts:i:30 RG:Z:control_2:0:1:H37KTDSX2:2 RE:A:I xf:i:0 CR:Z:TCGCTCAGTGGCTAGA CY:Z:FFFFFFFFFFFF:FFF CB:Z:TCGCTCAGTGGCTAGA-1 UR:Z:GATTTTTGGTTT UY:Z:FF,FFFF,FF:F UB:Z:GATTTTTGGTTT
A00814:412:H37KTDSX2:1:1547:6008:14215 16 chr1 3069555 3 90M * 0 0 TAGTGTCTGTATAACATCTATCCAGGCTCTTCTGGCTTTCATAGTCTCTGGTGAAAAGTCTGGTGTAATTCTGATAGGCCTGCCTTTATA FFF,FFFFFF:FFFFFFFFFFFFFFFFFF:FF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:2 HI:i:1 AS:i:88 nM:i:0 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:TACAACGCATGTTTGG CY:Z:FFFFFFFFFFFF,FFF CB:Z:TACAACGCATGTTTGG-1 UR:Z:TTCTTGTAAATG UY:Z:FFFFFFFFFFFF UB:Z:TTCTTGTAAATG
A00814:412:H37KTDSX2:1:1536:5810:23296 0 chr1 3085672 0 30S60M * 0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGCCCAACAATCAAAGAAAATGCAAAATGCAAAAAGATCCTAACTCAAAACATCCAGGAAAT FFFFFFF:FFFFFFFFFFFFFFFFF,FFFFF,FFFF:F:FFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF,FF NH:i:5 HI:i:1 AS:i:59 nM:i:0 ts:i:30 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:TGTAACGCAGTTCACA CY:Z:FFFFFFFFFF,FF:FF CB:Z:TGTAACGCAGTTCACA-1 UR:Z:TATGCGTCTCTT UY:Z:FFF::,FF:F:: UB:Z:TATGCGTCTCTT
A00814:412:H37KTDSX2:2:2449:17671:35947 16 chr1 3136662 0 90M * 0 0 CACCCCCACTCCCCTGCCCACCCACTCCCCCTTTTTGGCCCTGGTGTTCCCCTGTACTGGGGCTTATAAAGTTTGCAAGTCCAATGGGCC FFFF,FF:F:FFFFFF:FFFF:F,FFFFF::FF:FFF::FFF:FFF:F,,FFFFFF:FF:FF,,FFFF,F:FFFFFF,FFFFFF:FFF,, NH:i:7 HI:i:1 AS:i:86 nM:i:1 RG:Z:control_2:0:1:H37KTDSX2:2 RE:A:I xf:i:0 CR:Z:CAGGTATGTCAAGGCA CY:Z:FFFFFFFFFFFFFFFF CB:Z:CAGGTATGTCAAGGCA-1 UR:Z:CAAGAGCTATTA UY:Z:FFFFFFFFFFFF UB:Z:CAAGAGCTATTA
A00814:412:H37KTDSX2:1:2410:19298:14559 0 chr1 3142729 0 30S60M * 0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGACAGCATCCTAAATAACAAAATCAGAAATGAAAAGGGAGACATAACAACAGATCCTGAAG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:8 HI:i:1 AS:i:59 nM:i:0 ts:i:30 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:ACGTAACCACCCTTGT CY:Z:FFFFFFFFFFFFFFFF CB:Z:ACGTAACCACCCTTGT-1 UR:Z:TCTGGCTTCATC UY:Z:FFFFFFFFFFFF UB:Z:TCTGGCTTCATC
A00814:412:H37KTDSX2:2:2131:21151:7435 0 chr1 3142729 0 30S60M * 0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGACAGCATCCTAAATAACAAAATCAGAAATGAAAAGGGAGACATAACAACAGATCCTGAAG FFFFFFFF,FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFF:FFF NH:i:8 HI:i:1 AS:i:59 nM:i:0 ts:i:30 RG:Z:control_2:0:1:H37KTDSX2:2 RE:A:I xf:i:0 CR:Z:GTTACAGCAACCTAAC CY:Z:F,FF,FFFFFFFFFFF CB:Z:GTTACAGCAACCTAAC-1 UR:Z:CCTAGCCGACTC UY:Z:FF:FF:FFF:FF UB:Z:CCTAGCCGACTC
A00814:412:H37KTDSX2:1:2410:19533:14779 0 chr1 3142729 0 30S60M * 0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGACAGCATCCTAAATAACAAAATCAGAAATGAAAAGGGAGACATAACAACAGATCCTGAAG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:8 HI:i:1 AS:i:59 nM:i:0 ts:i:30 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:ACGTAACCACCCTTGT CY:Z:FFFFFFFFFFFFFFFF CB:Z:ACGTAACCACCCTTGT-1 UR:Z:TCTGGCTTCATC UY:Z:FFFFFFFFFFFF UB:Z:TCTGGCTTCATC
A00814:412:H37KTDSX2:1:2608:6614:7968 0 chr1 3154927 0 90M * 0 0 CCAGCTCCTATTTTAGCCACAAATCGTGGTGTTACTAATGACATAATTCTTGCCTAGGTCTTGCTAAATCTGAGGTTGATAATTCTCCTT FF:FFFFF,F,FFFFFFFFFFFFFFFFF:FF:FFFFFFFFFFFFFF:F:FFFFFFFF,FFFFFFFFFF,F:FFF,FFFFFFFFFFFFFFF NH:i:5 HI:i:1 AS:i:88 nM:i:0 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:CACGTTCAGTTTCAGC CY:Z:FFFF:FFFFFFFFF,, CB:Z:CACGTTCAGTTTCAGC-1 UR:Z:CTATGTACAACC UY:Z:::FF,FFFFFFF UB:Z:CTATGTACAACC
A00814:412:H37KTDSX2:1:1676:30653:8547 0 chr1 3156001 255 63M27S * 0 0 TCGGGTCCCCTCCCCCTTCCTTCATAACTAGTGTCGCAACAATAAAATTTGAGCCTTGATCCGAAAAAAAAAAAAAAAAAAAAAAAAAAA FFFFFFFFFFF::FFFFF:FFFFF,FFF,F:F::FFFFF:FFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:62 nM:i:0 pa:i:27 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:CTATCTAAGCCTATCA CY:Z:FFFFFFFFF::F,FFF CB:Z:CTATCTAAGCCTATCA-1 UR:Z:CCTCACCGATCG UY:Z:FFFFFFFFF,FF UB:Z:CCTCACCGATCG
A00814:412:H37KTDSX2:1:2675:31593:35634 0 chr1 3156001 255 63M27S * 0 0 TCGGGTCCCCTCCCCCTTCCTTCATAACTAGTGTCGCAACAATAAAATTTGAGCCTTGATCCGAAAAAAAAAAAAAAAAAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:62 nM:i:0 pa:i:27 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:CTATCTAAGCCTATCA CY:Z:FFFFFFFFFF:F,FFF CB:Z:CTATCTAAGCCTATCA-1 UR:Z:CCTCACCGATCG UY:Z:FFFFFFFFFFFF UB:Z:CCTCACCGATCG
A00814:412:H37KTDSX2:2:2625:17463:10786 0 chr1 3156408 1 21S69M * 0 0 TATCAACGCAGAGTACATGGGATGGAAGAGAGAATCTCAGGTGCAGAAGATTCCATAGAGAACATCGGCACAACAATCAAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFFFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:4 HI:i:1 AS:i:67 nM:i:0 ts:i:21 RG:Z:control_2:0:1:H37KTDSX2:2 RE:A:I xf:i:0 CR:Z:GTTTACTGTGGCAACA CY:Z:FFFFFFFFFFFFF,FF CB:Z:GTTTACTGTGGCAACA-1 UR:Z:GCAACTATAAAT UY:Z:FFFFFFFF:FFF UB:Z:GCAACTATAAAT
A00814:412:H37KTDSX2:1:1666:16712:7764 16 chr1 3163691 1 42S46M2S * 0 0 TTTTTTTTTTTTTTTTTGTGTTTTTTTTTATTTTATTTTTTTGGGTTTTTGTTTTGTTTTATTGGTTTTTTTATTTTTTTTTTTTTTTCG F,,FF:FF:FF:::F,:,F,::,:FFFF:,FFFF,:FFF,,FF,:,,F,:,:,,,F,F,,,,,,,FF,:F:,,FF:,F:,,::F,F,F,F NH:i:3 HI:i:3 AS:i:33 nM:i:6 pa:i:42 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:ATTCCCGCACAGAGCA CY:Z:FFFFFF:FFFF:FFFF CB:Z:ATTCCCGCACAGAGCA-1 UR:Z:AGATCATGTATA UY:Z:,FF:F:FFFFFF UB:Z:AGATCATGTATA
A00814:412:H37KTDSX2:2:2317:29532:19476 0 chr1 3168529 255 31S47M12S * 0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCCCGGGCCCGAGGG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFF:FFFF,:,,,,FF,,,,F,,,:FF NH:i:1 HI:i:1 AS:i:46 nM:i:0 ts:i:30 RG:Z:control_2:0:1:H37KTDSX2:2 RE:A:I xf:i:0 CR:Z:TTGATGGTCCCTAGGG CY:Z:FFFFFFFFFFFFFFFF CB:Z:TTGATGGTCCCTAGGG-1 UR:Z:TAACGTAACTTG UY:Z:FFF:FFFFFFFF UB:Z:TAACGTAACTTG
A00814:412:H37KTDSX2:1:1144:12689:27946 0 chr1 3168533 1 30S46M14S * 0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCGTTGTTAAAATAAAAATA FFFFF:FF:FFFFFFFFFF,FFFFFF:FF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,,F:,FF:,,,,:F,,FF,F:,FF,,F,F NH:i:4 HI:i:1 AS:i:43 nM:i:1 ts:i:30 RG:Z:control_2:0:1:H37KTDSX2:1 RE:A:I xf:i:0 CR:Z:ATGAGTCAGATACCAA CY:Z:FFFFFF,FFFFFFF:F CB:Z:ATGAGTCAGATACCAA-1 UR:Z:CGCCTTTCAGAT UY:Z:FFFF::F::,F, UB:Z:CGCCTTTCAGAT
Hi,
thanks for sending this out.
Indeed the CB:Z: should be there to indicate the CB field is a string. Without the Z, the bam file would not be read.
I think you are right and I had inadvertently broke something when I recently added the optional strand detection. I have pushed a new update which should fix the problem (at least it now works on my side).
Please let me know if you still have issues.
Best,
Hi there, I was trying to run your parseBAM.pl script on the cellranger bam of 10x single-cell short-read sequencing but the matrix outputs are all empty.. does your script need something unique from STAR's output or something else? Also if you could share more about how you process the 10x data it'd be appreciated, thank you!