SNP calling ONT sequenced files

Question

[blur]

Hi,

I have ONT files (fastq) and want to read SNPs on them. I have read some papers saying ONTs are too error-prone - is it still true?

Also, I have tried doing this on CLC workbench and couldn't - the fastq files were too big. The company support could not offer help. My sequenced isolates are bacterial - 3-6 million base genomes, so the large chromosomal contig is usually 3-6 million bases long.

Is there a good and reliable tool for ONT SNPs reading? Would it make sense to break the contigs into smaller portions or will that hurt the reliability of the results?

Thanks in advance

[GenoMax]

What is the median length of raw reads and how many do you have? Has the data been basecalled with "high" or "super" accuracy?

I have tried doing this on CLC workbench and couldn't - the fastq files were too big

If you have excessive coverage (> 50x), you could subsample the fastq data and see if CLC is able to work with that.

Answer 1

[colindaven]

The quality of (modern, SUP basecalled, but certainly not fast basecalled) ONT sequences should be fine, especially for bacterial SNP calling these days.

Have a look at deepvariant (resource-intensive) or longshot (simple) via bioconda to get started with SNP calling on long reads.

Answer 2

[cfos4698]

My personal preference is using clair3. I'm unsure if CLC didn't work because your compute resources are too limited, or if there are restraints placed on CLC workbench itself (e.g. RAM limiting). In any case, you'll likely need to use the command line for clair3 if you use it regardless. A simple workflow that you could expand on would be to QC/trim the reads with something like nanoq, map them to your chosen reference genome with minimap2, then call variants with clair3.