Choosing the Correct Trim Length

[macrobiome (T)]

I am new to bioinformatics, QIIME2, and any sort of coding/programming. I am currently trying to analyze some 16S data (PE-250 run) on QIIME2 v2023.2. The goal of my project is to observe and correlate the translocation of the microbiome from highly enriched regions (intestines) to regions with generally much lower numbers of bacteria (tumors).

After a lot of struggle, troubleshooting, and learning, I was able to reach the quality score stage, but I am getting conflicting information on choosing the correct trim length.

For the forward reads, I am thinking of keeping almost the entire length (maybe trimming it off at 250) and for the reverse reads, I am thinking of trimming it at about 225.

Do you guys have any input on this? Am I moving in the right direction?

Thank you so much!

[crusher083 (Valentyn Bezshapkin)]

Hello and welcome to

A general answer: it's hard to tell if you're doing everything fine without knowing exact commands. That said, there are only a few steps before this one, and QC looks adequate.

QC answer: there is no conflicting information, only subjective choice. Your cut-offs look adequate to me. I would also trim lower-quality first few bases using --p-trim-left-f and --p-trim-left-r.

Cheers,
Valentyn

[macrobiome (T)]

Thank you for your input, Valentyn!

This is the only command I ran:

qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' /
--input-path "/Users" /
--input-format CasavaOneEightSingleLanePerSampleDirFmt /
--output-path paired-end-demux.qza /

I plan to run the following command

qiime cutadapt trim-paired --i-demultiplexed-sequences paired-end-demux.qza /
  --p-cores 2  --p-front-f GTGCCAGCMGCCGCGGTAA --p-front-r GGACTACHVGGGTWTCTAAT /
  --p-quality-cutoff-3end 20 --p-quality-cutoff-5end 20  /
  --p-minimum-length 50 --o-trimmed-sequences demux-trimmed.qza /

After this, I will do denoising and truncating. Thanks!

[crusher083 (Valentyn Bezshapkin)]

Yes, it looks like the output is adequate for your commands.

Cheers
V