analysing nanopore amplicon data

Question

[khoojj]

Hi,

We are planning to sequence amplicons from 42 intron loci (from a multiplex PCR) for multiple samples (barcoded using ONT barcodes during library prep) on nanopore. We are interested to see if the 42 intron loci would be useful to differentiate multiple closely related rodent species. I wonder if there are tools similar to AmpliSAS (http://evobiolab.biol.amu.edu.pl/amplisat/index.php?amplisas) that has been used with nanopore data?

Any suggestion for a workflow available that is good for working with amplicon data for genotyping will be appreciated.

Thank you.

[dthorbur]

There are a few different methods you could use. AmpliSAS is a great tool, but is susceptible to false positives if you have a high error rate, like that associated with nanopore sequencing. You can reduce the number of false positives by sequencing technical replicates of each sample and only taking sequences found in both technical replicates - you see this in studies of hyper-polymorphic loci like MHC.

You can easily recreate each step of AmpliSAS with other tools. For example, you could cluster your sequences using something like UMAP and see if that agrees with your phylogeny.

Alternatively, you could use metagenomic tools like Kraken2 and a bespoke database of your species to map your reads and see what they resolution is. They are designed for 16s/18s rRNA amplicon sequencing which have started to use nanopore sequencing in some instances. See a 2016 benchmark paper here.