Determining the PHRED Quality Score

Question

[adhamzul]

Hello,

I am currently trying to set-up a de novo genome assembly, analysis and annotation system for my lab, using Oxford Nanopore Technology (ONT) MinION sequencing, for marine microorganisms. We are focusing on strains that might be new or not-yet-researched.

I have no experience in nanopore sequencing nor bioinformatics so I am very sorry if this comes off as naive or dumb.

I have been NanoQC to check the quality of the ends of reads, using NanoPlot and NanoFilt to check the reads and trim them to increase the quality of the reads. Then I proceed to assemble the draft assembly with Flye, polish the draft assembly with Medaka and check the consensus.fasta file for completeness using BUSCO.

But, I am having trouble to determine the minimum PHRED score cut-off point. The person that was doing this before me has graduated and it was determined that the minimum cut-off would be reads that are Q8 and below. But it was not specified why.

It would be helpful if someone can point me to any papers that can help me to determine an absolute minimum cut-off score. Also, if anyone can point me to any papers that specify in detail the steps and the reasoning behind de novo genome assembly steps.

Regards, Adham

Answer 1

[GenoMax]

Phred quality scores were originally developed for human genome project. There is no absolute recommended quality score cut-off. This is somewhat subjective and dependent on analyst using the scores. Are you referring to average Q scores over the entire read (when you say Q8) or is it just those bases where Q drops below 8 (or some windowed interval)? Generally one would want to restrict to higher Q scores (say Q15 and above) if you are truly doing de novo assemblies (i.e. no reference available).

[adhamzul]

Thank you, GenoMax for your reply!

I am sorry for not clarifying, but yes, I was referring to the overall average Q score of a read when I said Q8.

So for example, for Q15, I assume that would be referring to the average Q score of a read? Do you by any chance know of a paper that states the reasoning for choosing to trim reads below a certain Q score?

Again, I am very thankful for your reply.

Regards,

Adham