Using CRISPResso2 for edited wheat polyploid genome screening

[kawaiininji]

Hi all!

First things first: im crispy new to bioinformatics.

I would like to use CRISPResso2 to evaluate CRISPR-edited wheat plants, which have polyploid genome. I don't know how should i start to process my NGS fastq files. The online version can't handle this, so i installed it to Ubuntu - that is all i could do. My question: Where should i start? What main pipeline "steps" that are MUST for this kid of work?

If you have any recommendation, i would appreciate it! Im eager to learn more.

Thanks,
Sara

[kclem]

Hi @kawaiininji,

Thanks for considering CRISPResso. What problems are you having in getting the online version to work? Have you been able to install CRISPResso in Ubuntu?

There are a few examples to get you started here: https://docs.crispresso.com/suite/core/examples.html For your analysis of multiple homeologous copies you should specifically look at the Multiple Alleles example.

If you have fastq files, the easiest place to start is by running

CRISPResso -r1 {read 1 fastq file} -r2 {read 2 fastq file} --auto

This will automatically try to find the reference sequence and try to align all reads to that reference and give you a rough estimate of what happened in your editing experiment.

Next, you'll want to rerun CRISPResso but also provide the exact amplicon and guide sequences (--amplicon_seq and --guide_seq) If you know what your reference sequence is (the sequence between your primers) you can provide that to CRISPResso using the --amplicon_seq parameter. If you want to separate out the effect on different homeologous copies of the target gene, you can provide multiple amplicon sequences as a comma-separated list to --amplicon_seq. Providing the --guide_seq parameter will restrict classification of editing events to those at your guide cut site, and add guide annotations to your output as well as plots focused on activity at the guide. You can find the full list of parameters here: https://docs.crispresso.com/suite/core/parameters.html

I hope that helps, but feel free to reach out if you run into any more problems.