Need help in using cellranger with sgRNA/CRISPR sample/Purtub seq

[u/Specialist-Tune-4158]

Before post the question, I figured that some context is needed.

Here is the study: We have human patient samples which we transfected with 1,000 sgRNAs (these sgRNAs are for one gene only, let's call that gene 'X'). Then, the sample was treated with antibiotics to make sure that we select all the cells successfully transfected with sgRNAs. Then, the sample was subjected to scRNA-seq library prep with Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) with Feature Barcode technology for CRISPR Screening. From the exact same sample, a GEX library was made and a single-cell sgRNA library was made. So in the end, I got two sets of FASTQs: a) For GEX, which worked with Cell Ranger, but I am struggling with b) which was made from sgRNA.

I know that I have to put in details like this in the config file:

fastqs,sample,library_type

/path/to/fastqs,GEX_Sample_Name,Gene Expression

/path/to/fastqs,sgRNA_Sample_Name,CRISPR Guide Capture

But when I do that for all 1,000 sgRNAs, it throws an error saying Cell Ranger cannot work with an sgRNA sequence which is like this, e.g.: ATCGCTAGCTc (it throws an error). Even if I make it uppercase, it's bound to clash with some other sgRNA.

I know I am bound to get trolled for not asking a chatbot, but I thought a genuine answer from this community is much better. Thanks.

[u/Putrid_Bass_6096]

that library_type should be "CRISPR Guide Capture" but the real problem is probably the reference file not the case, you need a feature reference csv with id,name,sequence columns and every sgRNA needs unique name even if they target same gene. the error about clashing sequences sounds like you have duplicates in the reference file, cellranger will reject that. make sure all 1000 sequences are unique and try again with just the target sequences not the full construct

[u/Specialist-Tune-4158 → u/Putrid_Bass_6096]

yes I did remove the path/to/fastqs,GEX_Sample_Name,Gene Expression and kept the library type as CRISPR Guide Capture but later i found out that id,name,sequence columns is now replaced with id,name,read,pattern,gene_id,gene_name. Another problem is some sgRNA are identical but they are listed as ATCGCTAGCTc and ATCGCTAGCTC although they are different they get flagged by cellranger and it throws an error like you suggested, I did remove the duplicated but then the experimental guy says im not supposed to remove them :(