Help Analysing Perturb-Seq Data - How do I extract 10X cell barcodes and relevant guides from fastq files?
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4 weeks ago
jesswhitts • 0

Hello,

I am analysing some perturb seq data. For the single-cell, ECITE-seq was used, so we have GEX and protein expression values. The perturb-seq guide library was sequenced separately. I have managed to analyse the ECITE-seq part as normal, using cellranger and a CITE-seq workflow, but I'm not sure of the best way to integrate the perturb-seq guide information...

I think I need to somehow create a table containing the 10X cell barcode, 10X UMI, and alignment to a relevant sgRNA from the ones used. Any suggestions on how I can do this? I suppose I need to create a custom reference transcriptome for the sgRNA library and align my fastq files to it?

Best wishes, Jess

Single-Cell Perturb-Seq 10X • 504 views
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Entering edit mode
4 weeks ago

If you just want to extract relevant features like 10x barcode or UMI from fastq data you can process the fastq manually.

For example this will extract the 16-nt 10x barcode from a typical R1 by removing the last 10 characters from the 26-length read

cat Your_R1_001.fastq | \        #cat the R1 and pipe it to awk
awk '(NR%4==2)' | \              #take every 4th line of the fastq starting at line 2
sed 's/.\{10\}$//' > Your_R1.txt #empty the last 10 characters to only keep the 16-nt 10x barcode

If you know the structure of your R1/R2 then you can manipulate them as needed. But as you said, you probably should try a more sophisticated and robust approach

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26 days ago
jesswhitts • 0

Found a pretty simple solution in the end - using cellranger multi and rerunning my analysis with the CRISPR guides as features

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Good to know! Thanks for updating with a solution

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