I wrote a method to check CRISPR editing in transgenic plants with NGS, but that method needs a whole sequening lane, which is usually an overkill for CRISPR checking. We found MGH and Genewiz provide PCR amplicon sequencing service. Each sample is about $70, but we can mix our PCR amplicons with barcodes using the same two-round PCR method in my last post. Because the service provider will add sequencing adapters and their barcodes to the submitted samples, we need differnt adapters and barcodes this time. I have designed new PCR adapters and barcodes for PCR amplicon sequencing. You can download the protocol here and the barcodes here.
The results are fastq files and you can analyze by the following steps:
- Check quality and filtering with fastp here.
- Demultiplex the filtered fastq files here.
- Map reads to your PCR template and get SNP and small indel calls here with BWA-MEM and SAMTOOLS. You can also try other read mappers like strobeAlign and HISAT2, but the results should be very similar.
- Check large indels (>15bp) and structure variations here with Subread.
- View the downloaded bam files with IGV. You need to install IGV first. The web version does not work well.