Hi everyone, i have attached an interactive quality plot of my archaea and bacteria paired end sequencing and was hoping someone could confirm or suggest the trimming?
The trimming position depends on both the quality and on the region you are trying to amplify. After trimming, you still want to reads to overlap a bit so they can be merged, and this depends on the length of the region sequenced.
What region did you sequence for Bacteria and how long is it? What about for Archaea?
Hi Colin , Thanks for the reply. This is what i wasn't sure about. For Archaea it was the v6-v8 region.
For Bacteria it was v3-v4 region. Both of them on the PCR was around 500 bp.
Ah, I think this might cause a problem with the truncation-length you provided in your --p-trunc-len-* commands.
Based on the trimming discussion that Liz linked to you, how much overlap between archaea reads would expect after passing --p-trunc-len-f 120 and --p-trunc-len-r 120?
Ah, OK. That's good to know. Do you know the specific primers used? I ask because many primers are labeled with their location on the E. coli 16S gene. For example, the 515f and 806r primers start at 515 and end at 806, making an amplicon ~291 bp long.
I agree. Do you want to show me how you calculated the overlap/gap, and how long you would expect the gap to be after trimming at 220, and how long you expect the overlap to be if you did not trim at all?
That's right. 440 of coverage is 24 bp less than 464, resulting in a 24 bp gap.
DADA2 requires 12 bp of overlap by default. If you used the full forward read (250 bp long), where should you trim the reverse read to get 12 bp of overlap?
Thank you so much for your help Colin, it is running now and we will see how it looks. The DADA2 step was the hardest part for me to wrap my head around.