Hi,
I have a question related to the trunc-len value for the denoise step of DADA2. Please correct me if I am wrong, I have read that it is suggested to truncate sequences when the twenty-fifth percentile quality score drops below 25. Based on my quality plots this is going to be approximately 268 and 182 for forward and reverse respectively.
Will the 86 base difference hurt my output by introducing length inconsistencies?
Hi @Miri_J,
For choosing truncation length, I do not have a hard rule for where to truncate. I usually look for where there is a large dip in quality. So, personally I probably would choose truncation parameters around 280 and 200 hundred respectively. However, its really up to the researchers so I think your values would be fine, and the difference of the length shouldn't effect anything especially if you are joining the reads together.
The only thing is that you want to make sure there is 12 bp overlap between your forward and reverse reads. This can roughly be calculated by the forward length + reverse length >= amplicon length - 12.
thank you so much! I have been trying to understand why analysis files produced by sequencing center our lab is using are so different from mine. It looks like their filter is much less strict than the one I have been using so far, because I have been getting 70% of input passed (on average), whilst they got mid 80's%
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