Hi, everyone. I would like to ask about the amplicon sequence, or other related methods for sequencing of CRISPR/Cas mediated knockout plants. Our lab works on plant functional genomics and knockout multiple genes/alleles for hundreds of transgenic events, and need to sequence target genes to confirm gene mutation. We used to use the amplicon sequence by adding the barcode to PCR products which were amplified from genomic DNA. We shipped our samples to Eurofin and/or Azenta, however this method does not provide so good sequence results. Does anyone know better way for high throughput sequencing?? Any comment, suggestion and information will be appreciated. Thank you!!
Hi Michihito
amplifions are easy, suitable and reliable methods for NGS sequencing.
the important thing is to verify if your PCR targets are relevant to your crispr targets. you need to check, by hand on IGV or UCSC with custom tracks, or by command line using bedtools.
all the ebst
fred
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