Variant calling on Nanopore reads

Question

[felix]

I am currently working on nanopore reads, and trying to calculate the SNP and Indel count. I mapped the reads with Minimap2. From here I called the variants with GATK.

I know that GATK is actually written for short Illumina reads, so I was wondering if the results from the variant calling are trustworthy and whether there is a tool which is specialized on calling variants based on nanopore reads.

Thank you for your help

[ATpoint]

Also see Nanopore SNP and indels calling tool

[emilydolivo5]

Hi @felix , colindaven , @WouterDeCoster I'm a bioinformatics student currently delving into nanopore reads.

I would greatly appreciate it if you could share the steps and tools you've used for your work.

If you happen to have a GitHub repository related to this, sharing it with me would be fantastic. I'm eager to learn more about handling this type of data, and any guidance from your experience would be invaluable.

[colindaven]

@emilydolivo5

Something like this pipeline https://github.com/epi2me-labs/wf-alignment, or the commands contained within, should be useful for alignment.

If you have learned how to align reads to a reference before with minimap2, and use samtools to convert to bam then just do that. After you have a BAM longshot is one command.

Start with a conda env of those programs and see how far you get.

[emilydolivo5]

colindaven , Since my fastq files were generated using multiplex minion nanopore sequencing (long reads) applied on fungis does wf-alignment , sniffles2 and freebayes could be used in this situation ?

[colindaven]

Please make a new Post if you have a long series of questions about your project.

In general I recommend looking at the Galaxy training network for these questions

mapping reads https://training.galaxyproject.org/training-material/topics/sequence-analysis/tutorials/mapping/tutorial.html

calling variants https://training.galaxyproject.org/training-material/topics/variant-analysis/

They are very similar for nanopore, just use the different tools recommended here. You'll learn at lot if you do the tutorials first.

Answer 1

[WouterDeCoster]

I would suggest using Longshot.

Answer 2

[colindaven]

Clair also has a good reputation in addition to Longshot https://www.biorxiv.org/content/10.1101/2020.02.07.939322v1?ct=

However I have found Longshot https://github.com/pjedge/longshot quite easy to install and run, as long as you can get over the initial RUST hurdles. Fascinatingly, it's the first tool I've seen which seems to be able to handle long reads' ability to actually (easily) phase SNVs.