Mapping to custom reference minimap2/IGV

Question

[sendhelp]

I'm trying to map nanopore reads to a mtDNA reference using minimap2 for the purpose of determining how many mitochondrial reads are present in the nanopore data.

The minimap command I'm using is;

~/apps/minimap2/minimap2 -ax map-ont mtref.fa reads.fq  > aln.sam

I know there are mtDNA reads present as I can align them to the reference, however when I look at the resulting indexed and sorted sam file in IGV there's no coverage?

Thanks!

[GenoMax]

Have you made a custom genome in IGV using the same reference? By default IGV shows you the entire reference so you may need to zoom in before you start seeing the reads.

[sendhelp]

I have. I solved the initial problem by actually creating a sorted bam file and using that in IGV rather than just the same file. Thanks!

Answer 1

[ATpoint]

You can simply check the full alignment using samtools idxstats for the number of reads mapping to chrM.No need to align against a specific subset (which is also not recommended).

[sendhelp]

Thank you! Can I ask why it isn't recommended to align against a subset (I think you're referring to aligning genomic reads to a single chromosome reference?)

[ATpoint]

The aligner will always try to find the best match for each read in the library. If your library contains reads from the entire genome but you include only one chromosome (or one region) with the true origin of the read missing the aligner might false-positively put it somewhere else. I never quantified what the extend of false-positives is but this opinion is pretty much what I read here at Biostars from many of the more experienced folks.

[sendhelp]

Ah, makes sense! There isn't a reference genome available for me currently so I guess this is the only method?

[ATpoint]

Well I am spoiled by working with mouse and human only (and I often forget that when writing answers :-D ) but yeah if you have no reference I would include as much of the draft genome or the contigs you have and then filter for the regions/contigs/chromosomes you need.