Best tool for trimming and filtering nanopore direct RNA sequencing.

Question

[kenneditodd]

Hello,

I am new to analyzing long read data. I am trying to create a pipeline for analyzing data from Oxford Nanopore's MinION Mk1b using the R.9.4.1 flow cell and using the Direct RNA Sequencing Kit. We used only one sample - a mouse brain for practice. I have been reading about tools used for filtering and/or trimming reads. As a starting approach I wanted to trim adapter sequence (RTA, RMX) and filter based on average read quality. However, I am not sure on which tool to use and the minimum thresholds to apply. Additionally, does adapter trimming need to be done if I have used MinKNOW/Guppy to basecall?

Here are some of the tools of have read about: BoostNano (unsure if this checks RMA/RMX adapter content, fast5 as input), Pychopper v2 (cDNA only), Porechop (no longer maintained), Chopper (doesn't filter adapter content only contaminant DNA, part of NanoPack), and ProwlerTrimmer (was looking for something more simplistic with end trimming). Please let me know if my assessment is wrong of one of these tools.

Answer 1

[cfos4698]

I can't speak for the 'best' tool, but for our ONT sequencing we carry out adapter trimming with guppy, followed by further quality trimming/fitlering with nanoq (https://github.com/esteinig/nanoq).

Additionally, does adapter trimming need to be done if I have used MinKNOW/Guppy to basecall

Depends how you set up the guppy_basecaller analysis. In MinKNOW you should be able to specify a bunch of parameters, including whether to carry out adapter trimming. If you did not set these up in MinKNOW, you can still adapter trim after MinKNOW by running guppy_basecaller manually. IIRC, guppy_barcoder can also adapter trim, but don't quote me on that (I don't have barcoder on the computer I'm using at the moment).

[kenneditodd]

Thank you! This was helpful.