Nanopore data analysis

Question

[sarahmanderni]

Hi, I have Nanopore data, and the goal is to identify differentially methylated regions. I already have aligned BAM files with MM/ML tags. Does this approach make sense:

Convert BAMs to BED using modbam2bed

Aggregate % methylation (e.g., with bedtools)

Compare conditions using DSS in R?

Also, is it common to filter bam_pass reads by quality before analysis? And since I have ~500 BAM files per sample (about 150 GB total), should I merge them before converting to BED? Thanks in advance!

[GenoMax]

How about using modkit provided by Nanopore for methylation data analysis: https://github.com/nanoporetech/modkit

[sarahmanderni]

The overall size for all bam files is around 150 GB. Is this common for nanopore and does this tools handle this volume of data? Thanks for the help!

[GenoMax]

Data volume is going to depend on the amount of sequencing. Was this a PromethION flowcell? Tool should handle the data as long as you have adequate infrastructure available to process.

Answer 1

[colindaven]

• Merge bams - yes. (why do you have so many ?). Merging fastqs before creating bams is easier (cat *.fastq.gz ) than bam merging.
• 150gb - yes, typical
• modkit should be able to handle that amount of data, just try it. It's certainly more up to date than modbamtobed