Tips in trimming and truncation length

[RCM (richardM)]

Good day!

I am new to QIIME2-2019.1

I am stuck with the denoising stuff. I am not sure about the trim/trunc length. I have read multiple forum yet I couldn't decide the parameters. I am currently doing 16S V3-V4 region of MiSeq data. Any tips? Thank you.

[ebolyen (Evan Bolyen)]

Hi @RCM,

Wow, those are some really nice quality plots! I’d say your sequencing run went very well.

Which is really good since the V3-V4 region is fairly long and can be hard to hit (depending on your specific primers I think you only have about ~90 bases free after 2x300 sequencing for that region).

Your forward reads are excellent, I would probably keep the entire length or trim at ~280 (use trunc-len of 0 to skip truncation in that direction).

You reverse is also great, I would probably trim around ~270, but you have enough wiggle room that you could trim a little closer like ~240 if you kep the full length on the forward reads.

Then see if you get good merging from your denoising stats. Ideally you won’t see much/any drop-off at that point, but let us know if the counts before/after merging don’t look good.

[ebolyen (Evan Bolyen)]

Oh and make sure you trim off your forward primer’s length with trim-left. That may cover the slightly lower quality on the start of the read (which is very normal), or if your lower quality is a bit longer, go ahead and extend the trim-left a few more bases.