We have prepared a library of a selected gene by performing error prone PCR. We are interested in identifying the single nucleotide variants (SNVs) in the library and their frequency. Since the gene of interest is larger than 500bps ( it's 800 bp) and we don't wan't to do fragmentation, we can't use ILLUMINA Services. I was wondering if nanopore sequencing would be a good option for this goal. and if so, what is the minimum coverage you recommend ? Thanks in advance.
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Meghan.T •
Using Nanopore would be a good option though you have to keep in mind that the error rate will be slightly higher than illumina. If you have only one gene then a single MinION run should be adequate. You should use "super accurate" basecalling and then you can use error correction.
Nanopore has a workflow for this available: https://github.com/epi2me-labs/wf-amplicon
Thank you so much @GenoMax . Can you advise me on the basecalling tools for nanopore? I believe the company sends the fastq files but I might also be able to get the raw signal files as well.
Thanks! As I understand this workflow is for variant calling, right? I was wondering if you could kindly suggest some tools for basecalling as well.
Now dorado is the default basecaller for MinKNOW. https://github.com/nanoporetech/dorado
It is performant on the right GPU but in a pinch will work on CPU's as well. You can use it off-line to do SUP (super accuracy) basecalling.
Awesome thanks a lot!
You'll get massive coverage for just one gene (maybe 2000+ depending on how long you run the minion for), so you could consider multiple genes, or if you can get them using flongle flow cells.
SNP callers - longshot is probably the easiest.
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