Merge, Split, and Analyze with CRISPResso2

[modifiedplant]

To reduce cost, our lab uses combinatorial dual indices (HiTOM primers) to multiplex NGS reactions. The pipeline looks something like this:

Merge paired-end reads: FLASh
Demultiplex/split based on L and R barcodes: CRISPRMatch
Analyze: CRISPRessoBatch

I am wondering if the demultiplexing can be performed in CRISPResso so that the pipeline can be consolidated into a single tool. Ideally, one would supply a .csv file containing the sample name, barcode_L, and barcode_R. Then, Batch mode can be used to analyze the demultiplexed samples. I see that CRISPRessoPooled is available as well: will that work for this application? The barcodes are short (4bp) near each amplicon terminus. It is not uncommon for us to demultiplex paired-end NGS results into 50+ fastq files after merging.

Thanks in advance!

[kclem]

Hi @modifiedplant,

We could definitely look into this if there is a demand by the broader audience. Do your L and R barcodes define specific samples which have amplicon sequencing at the same locus? Or what are you barcoding?

[modifiedplant]

Hi @kclem,

Thanks for your response. The L and R barcodes indeed define specific samples. The amplicons are typically at the same target region. There is certainly a demand from a broader audience, from the Plant Science community, in particular. For example, this allows us to perform deep amplicon sequencing on many transgenic plants in a single NGS reaction.

If you'd like to connect about this with further details, let me know, and I can send you an email. Thanks!

[kclem]

Yeah - happy to connect. You can email me at [email protected]