Nanopolish with biological replicates

[lthomp06]

Hello,

I am looking to identify differences in methylation between two isogenic replicates. I have oxford nanopore sequencing of three biological replicates for two strains, and am looking to compare methylation between them. I have followed the quickstart tutorial on methylation calling, however, it seems the assigned positions are difficult to compare between replicates as the chromosome position varies slightly.

I would much appreciate any advice on how to compare these samples with the replicates. Thank you kindly, Laura

[jts]

Hi Laura,

The chromosome positions should not vary, can you give an example of the problem?

Jared

[jts]

Maybe I misunderstood, are you using two difference reference genomes?

[lthomp06]

Hi Dr. Simpson, I've attached a screenshot here of hits with methylation frequency of 1, if the chromosome position shouldn't change then I guess this is indicative of biological variability? I ran nanopolish with the same reference genome for all of these samples Laura

[jts]

In this screenshot the sites are fairly low coverage (only a few reads covers each site) so it is hard to say whether it is biological variability or just sampling noise. You should try to gather 20-30x coverage and only analyse sites that are covered sufficiently well.

I hope that helps!

[lthomp06]

Hi Dr. Simpson,

Thank you for your help interpreting the output file, it has helped immensely. I was wondering if you could recommend any methods to analyze differences in CpG methylation between two strains using the nanopolish output file? Currently I have been plotting methylation frequency using ggplot in R, although I am wondering if there are any more quantitative way to identify differences.

Thanks, Laura

[jts]

Hi Laura,

Unfortunately I don't have a strong recommendation here, as our focus is on the detection of methylation rather than downstream analysis. We've played around a little with DSS but I'm not confident enough to provide detailed advice.