Questions on using BAM file generated by 10X Cellranger

[pariaaliour]

Dear @yfarjoun,
Thanks for the very helpful tools!
I am trying to get the splice junctions out of single cell RNA-seq (10X) dataset as below:

regtools junctions extract -b output.barcodes -o output.sj possorted_genome_bam.bam -s XS
I used the BAM file generated by 10X Cellranger, and here is the information of the first read for your reference:

A01433:57:HK7V5DSX2:2:1219:31729:12602 16 chr1 10006 1 101M * 0 0 CTATTCCTTACCATTAACCTTAACCTTACCCTTACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAAC ,:,,,::,F:,:,:::,,,,:,,:,:,F::FF,F:,,,F,::FFF,:F:F:F,F,FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:3 HI:i:2 AS:i:79 nM:i:10 RG:Z:T01556_sl_con:0:1:HK7V5DSX2:2 RE:A:I xf:i:0 CR:Z:ATCCTATGTTGACGGA CY:Z:,FFFFFFFFFF,FFF: CB:Z:ATCCTATGTTGACGGA-1 UR:Z:TTCAGCAGGGCT UY:Z:FFFFFFFFFFFF UB:Z:TTCAGCAGGGCT

I have some questions as below:
First, do I need to specify the PCR adaptors (cell barcode and UMI) in output.barcodes? or it is sth that it find through the bam file as if I am not proving it I get the warning: No CB tag found for alignment (id = 0) . And does not providing this make any difference to the output?

Second, I was wondering if the format of my BAM file (in terms of tags) is appropriate to run the above command?

Thank you for your help on this!
Paria

[yfarjoun]

Hello @pariaaliour,

I'm not sure if this is the best place to get answers for your question.

Personally, I'm not one of the maintainers of regtools and so I would refrain from trying to answer your question here.

If the team here doesn't help, perhaps if you get better results on https://bioinformatics.stackexchange.com/