Hi everyone,
I am usually accustomed to perform quality trimming on 16S-targeted MiSeq PE reads prior to merging them, however my sequencing facility suggested that they had obtained very good results by quality trimming after merging the reads.
In a way this makes sense. In the good old days of Sanger sequencing, we would combine paired reads into a contig which would be a quality check in itself. We would then quality check and trim the merged reads.
Has anyone been performing the quality trimming after read merging? Does anyone know how this choice may impact the data down the line?
All inputs will be greatly appreciated!
Cheers,
Chris
There are many statements in your post that could deserve a bit more detail: what does your facility mean by good results? How do you/your facility does the merge? How do you/your facility does quality trimming?
Usually paired reads have poorer qualities in the ends, which usually correspond to the area that is to be merged. So, to improve the chances of a proper merge, you should remove incorrect bases from the reads. The downside of this is if you're too strict, you'll loose many sequences because they'll become too short.
What may get you better results is not to do quality trim and do the filtering in the merge phase, where you can more flexibly specify your criteria to accept merging. This way you keep longer reads, and check for the quality by looking for mismatches (as you mention for the sanger). What I usually do is with my MiSeq data is to perform an initial not very strict trim (remove bases lower than Q10 or Q20 from the ends of reads) to remove "obvious trash" and then filter in the merge phase.
Performing quality trimming after merging I think is not very likely to make much, but it depends on what you do for trimming (I usually trim from the ends of reads).
Just my 2c,
Daniel
We are also planning to do trimming after merging data. I think it's fine.
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