Recommendation for computational tools to detect gene editing induced INDELs from amplicon NGS data

Question

[lhuang7]

I am looking for a software program to detect INDELs in zinc-finger nucleases (ZFNs)-edited genes using amplicon sequencing data. The purpose is to evaluate the gene-editing efficiency.

After hours of literature search, I found most of the current tools are designed for CRISPR/Cas9 system, not for ZFNs or TALENs, e.g. CRISPResso2, CRISPR-DAV, CRISPR RGEN Tools, CrispRVariants, amplican, etc.

I was wondering if anyone is aware of tools designed for gene-editing system other than CRISPR/Cas9, or if it's possible to modify the current CRISPR/Cas9-based tools for amplicon sequencing data obtained from genes edited by other programmable nucleases such as ZFNs.

Thanks in advance!

Lei

[MSRS]

Hi, do you have NGS like illumina sequencing data?

If yes, you can call variant with respect to reference with tools like snippy. It will assist you to find INDEL.

[MSRS]

Hi, do you have NGS like illumina sequencing data?

If yes, you can call variant with respect to reference with tools like snippy. It will assist you to find INDEL.

Answer 1

[francois]

I have never used ZFNs but I routinely use ampliCan on Illumina MiSeq data for my Cas9-injected zebrafish embryos. I imagine ZFNs also create indels around an expected site? Then I don't see why ampliCan would not work. In fact, there is a bit in ampliCan's FAQ about using it with other genome-editing techs: https://rdrr.io/bioc/amplican/f/vignettes/amplicanFAQ.Rmd.

Note: typical variant caller (like snippy, I imagine) won't work in your case – been there done that! The reason is that they (at least the tools I know) don't expect to find many different mutations at the same position. The data is too messy for them, basically. I think you're looking in the right direction, you'll just need to invest some time in getting one of these tools to process your data correctly. I'd recommend always comparing what these tools tell you with what you see by eye in IGV. It's not an easy task to quantify this mess and they can get stuff wrong.