Question: scRNAseq: Split FASTQ into cell specific FASTQ Files
2
gravatar for niklas.lang
16 months ago by
niklas.lang20
niklas.lang20 wrote:

Hi ! I did scRNAseq (10XChromium) of cancer cells and now I am looking for a simple way to split my FASTQ Files into celll specific FASTQ Files. Alternativley split my BAM File into cell specific BAM Files - because I need one FASTQ (BAM) File per cell in order to do perform variant calling on the single cell level. My goal is to obtain one single VCF file per cell.

Is there a build-in function in CellRanger for that? Are there any other tools to demultiplex my FASTQ/BAM Files using cell barcodes and split them into cell specific FASTQ/BAM Files?

Any help is highly appreciated! Thank you so much!

ADD COMMENTlink modified 14 months ago by igor11k • written 16 months ago by niklas.lang20
1

It is incredibly unlikely that this will prove useful. 10X reads will be enriched around the polyA site and be highly redundant.

ADD REPLYlink written 16 months ago by Devon Ryan96k

My idea was to split the BAM File into several cell-specific BAM File by using the "CB" Tag. I don't know how this could possibly interfer the PolyA enrichment, so I would be glad to hear what your concerns are?

ADD REPLYlink written 16 months ago by niklas.lang20

The question is whether single cell variant calling with this type of data is likely to be useful. The answer is "no", because you're mostly sequencing around the TES.

ADD REPLYlink written 16 months ago by Devon Ryan96k

...meaning you're limited to detecting (exonic) variants close to the TES

ADD REPLYlink written 14 months ago by Friederike6.2k
3
gravatar for igor
14 months ago by
igor11k
United States
igor11k wrote:

If you want to call variants from 10x data, there is a specialized tool by 10x that is optimized for their data and uses the combined BAM file (no need to modify your BAM file): https://github.com/10XGenomics/vartrix

If you are still interested in splitting a BAM file by barcode, there are some approaches outlined in this blog post: https://divingintogeneticsandgenomics.rbind.io/post/split-a-10xscatac-bam-file-by-cluster/

ADD COMMENTlink modified 14 months ago • written 14 months ago by igor11k
1
gravatar for swbarnes2
14 months ago by
swbarnes28.6k
United States
swbarnes28.6k wrote:

You could split on the CB tag, but coverage in single cell datasets is so poor, you aren't going to find high quality variants.

ADD COMMENTlink written 14 months ago by swbarnes28.6k
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