Filtering low quality bases

[MNMoller]

Hi,

I would like to filter out or mask bases in my SAM file that has a quality value below a defined number. Is there a way to do this?

Alternatively I want to filter out the bases or mask them in my FASTQ file, but this has to be done without disturbing the BWA-MEM alignment, which I intend to do afterwards. This could possibly be done by replacing the law quality base call with a "N".

I am aware of quality trimming and removing reads by MAPQ, but this will not remove low quality bases within the read.

I would appreciate any input!

[finswimmer]

Hello MNMoller,

could you please describe why you want to do this?

fin swimmer

[MNMoller]

Yes, and this may require that I explain a little bit about my setup.

I am working on determining the fidelity of different enzymes. Therefore I have amplified a 200 bp target in a PCR and then sequenced it and counted the mistakes. I want to count the mistakes made by the enzyme and not the method. If I can get rid of the low quality base calls then I am also more sure that the basecall made by the instrument is correct. I have tried to overcome this by measuring background of the sequencing, which in theory should be sufficient.
However when quality trimming my read (from both ends), my results start to make more sense. So I guess the bottom line is that I want to test if a "quality trimming" within the sequence, will help the results make even more sense.