Low quality fast5 files

1

Entering edit mode

2.5 years ago

BPors ▴ 60

Hi,

I performed direct RNA Seq (with polyA) to my short sequences (~90 bp). However, my fastq_pass are only 20% of the total reads.

I used this quality script to do analysis for the quality score: https://gist.github.com/wdecoster/7cad6080950fa1e3ae3eaeeac4f6ae4d#file-perbasesequencecontentandquality-py

enter image description here

Do you have experience/thoughts for why I am observing such fluctuating qualities through the read body?

I appreciate your feedback. Thanks!

fast5 basequality fastq nanopore • 759 views

ADD COMMENT • link updated 2.5 years ago by colindaven 6.4k • written 2.5 years ago by BPors ▴ 60

1

Entering edit mode

I don't have to point this out for you, but your reads are very short. It may be that the basecaller isn't properly estimating the quality. I would suggest, if possible, aligning the data to a reference genome and looking at the percent identity of your reads.

ADD REPLY • link 2.5 years ago by WouterDeCoster 47k

0

Entering edit mode

Are you sure you set the right basecaller models for guppy? I messed this up once, and used the DNA model not knowing the reads were direct RNA, and got a load of rubbish out (my bad).

ADD REPLY • link 2.5 years ago by colindaven 6.4k

Login before adding your answer.