Is it possible to create a pseudobam file with kallistobustools (kb)?

[ahmadalajami]

Hello again,

I am running kallistobustools (kb) on BDWTA dataset (mainly, kb ref and kb count). I was wondering if it is possible to obtain a pseudobam file similar to the one obtained by kallisto quant --pseudobam? Thanks!

[Yenaled]

We do have a --genomebam in the devel version of kb that's fully functional (and it will be added into the next stable release of kb).

See pachterlab/kb_python#184

[ahmadalajami]

Perfect, I will have a look. Thanks @Yenaled!

[ahmadalajami]

Hi @Yenaled,

I think I have a problem with the BAM file generated.

I ran kb ref and kb count with Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz and Homo_sapiens.GRCh38.108.gtf.gz - downloaded from Ensembl - on a BDWTA sample with R1 and R2 specified.

However, the BAM file generated has seemingly weird flags. If I run samtools view pseudoalignments.bam | awk -F '\t' '{print $2}' | sort | uniq -c | sort -nr, I get the following:

833123557 272 
832963405 256 
129320471 0 
109541085 4 
24235875 16

None of these flags (272, 256, 0, 4, 16) is a paired read. Is it, by default, quantifying single-end reads? What is it that I could be doing wrong? I can't seem to figure it out just yet...

[Yenaled]

BD Rhapsody is single-end (paired-end doesn't mean you have an R1 and R2, it means you have two sequences on a fragment that are sequenced together).

[ahmadalajami]

I swear sometimes my brain stops functioning...
Sorry and as always, thanks a lot!