Extracting RNA sequence - single cell

Question

[Skamboj]

I am trying to extract the exact RNA sequence within the fastq file of a single cell dataset generated using Parse Bioscience. Is there a way to do this using star or something else, while completely ignoring the barcode data

I've created a reference that only has my gene sequence using bedtools. I would like to align my data to this fa file.

I was hoping I could just use bwa-mem2 and just do bwa-mem2 mem gene.fa r1.fastq r2.fastq > r.sam. Then, extract the sequence, but I am not sure if this will work

Answer 1

[dsull]

Only one of the FASTQ files (the r2.fastq file) has the barcode -- just use the r1.fastq file. In that way, your barcode sequence will be completely ignored.

Then you can run whatever (bwa, grep, etc.) against that r1.fastq file to find or align a sequence that you're interested in.

[Skamboj]

I started running alignment the other day using both files. Do you think the barcode info will cause any issues or will star and bwa just treat it as any other gene sequence that will go unmapped if it doesn't align to anything.

Even on the cluster the alignment process takes a few days, and I would rather not start over if I don't have to.