Hi all, I already have taken a few suggestions on my data and making a custom reference from particular genomic locations and Suggestions worked great. Now when I am mapping my ONT reads to this custom reference I am getting like 10 to 15% reads mapped to it. I understand that mapping stats would probably go lower as now there are no off target regions in custom reference but my reads are amplified for those custom locations so percentage should not be this bad. I used minimap2 and bwa mem with different settings but it didn’t work. If I do get better alignment with changed parameters then mapping quality becomes really worse .
I tried bbmap too but same result. Can someone please suggest me alignment tools or settings which would work better with custom reference and ONT MinION reads? p.s- I have some locations in custom reference which are like1 or 2 bp long( in case this is contributing in poor alignment?)
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vkgaur25 •
GraphMap is an option that OP can try, but it doesn't look like it's actively maintained and has been replaced by Heng Li's minimap2 in most pipelines.
Minimap2 or Canu:
But Canu is an assembler, no?
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minimap2 is generally the best aligner for long read sequencing data including nanopore, so I would suggest figuring out why you get such low alignment stats, rather than looking for an alternative tool.
Did you specifically try the program
mapPacBio.shfrom BBtools that is meant for long reads. You can't use defaultbbmap.shfor this type of data.No I didn’t use default one. I used mappacbio.sh only
Since you seem to have tried all good aligners (and not seem to be getting reasonable results) have you considered the possibility that the data in question may have some problems with it?
Why are you using a custom reference? Could you elaborate on your experimental setup?