Low quality fast5 files
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5 weeks ago
BPors ▴ 50

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

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

fast5 basequality fastq nanopore • 206 views
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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.

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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).