Pooled analysis

[AliaksandraR]

Hello,

I am having trouble with pooled analysis. The problem is with the structure of the amplicon file, specifically the header structure is not recognised.
What header structure is required?

Another question, is it possible to introduce additional parameters like trimming and quality filtering in the Pooled analysis?

Thank you very much for advice!

Best,
Aliaksandra

[Colelyman]

Hi Aliaksandra,

Thank you for using CRISPResso2 and I'm sorry to hear that you are having trouble structuring the amplicon file. Would you mind sharing the amplicon file and providing the version of CRISPResso you have installed?

Thank you,
Cole

[AliaksandraR]

Thank you very much for getting back to me!

I am using CRISPResso version 2.2.12. I've tried two different things. First, using a simple NHEJ set up. The amplicon file for that is attached. Using this, I get 3 warnings which are the same (Unable to find matches for header values. Using the default header values and order.), but the analysis proceeds and finishes. Second, I tried to use the prime editing mode, which gives the error message (Unable to find matches for header values. Using the default header values and order. ERROR: Incorrect number of columns provided without header.) and doesn't start the analysis. The amplicon file for that is also attached. I tried with no header or with a header, and got the same error.
I would appreciate any advice on this.

Also, I wanted to ask if it is possible to add the arguments from the basic CRISPResso in the CRISPResso Pooled suite? I.e., trimmomatic, quantification window, minimum read quality?

I also wanted to say that I am a recent user but really fell in love with the basic CRISPResso suite. It was very straightforward to install and learn how to use, even though I am not a bioinformatician.
Best wishes,
Alex
amplicon_info_nhej.txt
amplicon_info_PE.txt
amplicon_info_PE_header.txt

[Colelyman]

Hi Alex,

Thank you for providing the files, that definitely helps us diagnose what is going on. First, everything looks good for the NHEJ setup because when there are no headers the default order is used (name, amplicon sequence, and guide sequence). Note: if you don't provide headers and are curious about the order that CRISPResso is using you can add the --debug flag and it will print out the order of the headers that it inferred.

For the PE run, the file with headers seems to work on my end. Would you mind posting the full output (with the added --debug flag) when you run the PE file with headers? The PE file without headers unfortunately will not work because it is too difficult to infer between the different PE parameters.

As for adding arguments from the basic CRISPResso, yes you can definitely do that! You can just pass the parameters when you run the command, like CRISPRessoPooled ... -w 5 --trimmomatic_command /my/path/to/trimmomatic.

Very glad to hear that you had an easy time installing and using CRISPResso, thanks for the kind works!

Thanks,
Cole