Pre-Made sgRNA library FASTA file error

[smart0611]

Hi Jan:

I downloaded pre-made sgRNA library FASTA file (Sabatini lab; PMID 26472758) from the Help section. What is the regular expression I should use? I chose the first regular expression option on the dropdown menu (Standard for TKO, Gecko, Brunello) and it seemed it worked because I saw genes were blue and sgRNA were green on the right side. But when I hit 'Check and Upload', an error message comes back saying sgRNA identifiers are not unique. Is it because the FASTA file does not include specific number for each guide for the same gene?

I'd really appreciate your help!

Moonjung

[jwinter6]

HI Moonjung,

sorry for the issue with the FASTA file, I forgot to update the links in the help section :(

Please try the attached file.
As regex, just use the standard one (which is for lenticrisp).

Let me know if it worked :)

Cheers
Jan
FASTA_Wang_v2__185K_library_short_oligos_final.fasta.zip

[jwinter6]

Hi Moonjung,
did it work?

[smart0611]

Hi Jan:

Sorry for late reply. The new FASTA file you posted seems to be working as it went to the next step without error message. But I get 0 count for every sgRNA in my sample. This is likely due to the fact that my sequencing reads are only 20bp long (sgRNA sequence itself), so I used (.{20}) as regular expression. In theory, would 20bp reads still work for CRISPR AnalyzeR? Or some adaptor sequence is absolutely necessary to extract sgRNA sequence?

Thanks,

Moonjung

[jwinter6]

Hi,

have you tried to turn off the FAST FASTQ Extraction mode on the bottom right? This sometimes causes issues, my collegue is getting that one fixed.

cheers
Jan

[smart0611]

Dear Jan:

Yes, I did try to turn off the FAST FASTQ Extraction mode, but it still gave me "0" counts.

Thanks,

Moonjung