Filter reads from fastq files (10x) with a specific cell barcode

Question

[paulklein05]

Hello everyone,

I am analyzing perturb-seq data (10x). Unless cells were FACS sorted in the first hand, many of them (~40%) have not been associated to any sgRNA by cellranger. I observed that the BFP expression of the cells with no guide associated is comparable to other cells, showing that a CRISPR guide might be present but not detected / filtered out by cellranger ?

I would like to dig into this sgRNA attribution from the fastq files. To start, I would like to: 1) Select reads of a specific cell barcode (I chose a cell that has no guide according to cellranger, and a high depth) from the fastq file 2) Align these reads to a reference of all my sgRNAs (using bowtie2 ?) allowing 1 mismatch/indel

Do you have any recommandations for step 1 or 2 ? Would you suggest doing something else (I am very new to bioinformatics) ?

Best, Paul

Answer 1

[GenoMax]

You can use subset-bam program provided by 10x: https://kb.10xgenomics.com/hc/en-us/articles/360022448251-How-to-filter-the-BAM-file-produced-by-10x-pipelines-with-a-list-of-barcodes-

Cellranger includes unmapped reads: https://kb.10xgenomics.com/hc/en-us/articles/360004689632-How-do-I-identify-the-unmapped-reads-in-my-Cell-Ranger-or-Long-Ranger-output-

[paulklein05]

Thanks for the suggestions GenoMax :)