Good to know! Thanks for updating with a solution
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
2 answers
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
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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excuse me, can you share how to use cellranger multi? Especially the config csv. Thanks!
Please read the CellRanger documentation, there is no point to repeat what they have written there unless you ask a specific question.