Plenty of gaps with my sam alignment file, why is that?

Question

[daniel.castanedamogo]

Hello!

I am currently sequencing some a couple of malaria gene markers (msp2 and cpmp) across different patient samples. For that I am using nanopore MINion. One of my steps includes alignment to references, however I am testing my controls first (specifically a plasmid). Nevertheless, there is a lot of gaps and some regions have a higher coverage than others (see attached)! I was wondering if you guys could tell me the reasons behind it, could it be simply because how the library prep was done? Or the PCR was not amplifying the whole plasmid? My plasmid is about 6.5kb long and I used Minimap2 to align my reads to my reference. Also, I used tablet to visualize the sam file.

Thanks in advance!

https://ibb.co/WvFPG97

[Ram]

Please see How to add images to a Biostars post to add your images properly. You need the direct link to the image, not the link to the webpage that has the image embedded (which is what you have used here)

Answer 1

[WouterDeCoster]

I don't see any real gaps. What I can see are small false indels, which is the dominant error pattern in nanopore sequencing, especially in homopolymers. I don't know how you designed your amplicons, but unequal pcr amplification happens frequently and lead to uneven coverage.