What is the minimum read lenght to filter in modern samples?

Question

[lud_0911]

Hi,

I have 2x150 pb NovaSeq 6000 platform reads. I am familiarized to work with ancient DNA, but now I have to work with modern data. I used the cutadapt to filter out reads with min lenght of 35 bp and quality 30. Should I have used different values? What would be the difference if I use 80 bp for the minimum read lenght? By now, does someone has papers that support the values to use in modern data?

Thanks so much.

Answer 1

[GenoMax]

If you are aligning to a reference then no trimming, both based on quality and length, should be needed. Aligner should take care of clipping parts of reads that don't align. That said, if you have 150 bp reads and you end up with reads of length < 35 bp after trimming then you may have bad libraries (short inserts) or crappy sequencing (poor Q scores) (for normal DNA samples). Trimming at Q30 is really strict and should only be needed for de novo work.

[lud_0911]

Thanks for your answer GenoMax. So you are saying that I don´t need to be worry with the reads <35 bp in lenght because they won't pass in quality? Because I did my analysis filtering reads <35 bp, not <80 bp (a longer lenght for example). Should I redo with longer reads only?

[GenoMax]

What I was saying was that with normal (non-ancient DNA) data you should not have reads getting trimmed down to < 35 bp unless you have extremely short inserts or have very poor quality sequencing (which would lead to a large part of the read being trimmed off using your trimming criteria).

In practice, you can get away without trimming if you are aligning to a good reference. Most aligners will soft-clip reads as needed.