Get only v4 region from v3-v4 reads

[asbarros (André Barros)]

Hey everyone,

I have reads from V3-V4 region of 16s and was interested in extracting only the V4 region out of my reads.

Searched the forum and found this Q&A:

Extracting V4 region from V3-V4 data

Hi I have two different 16S rRNA gene data sets of the microbiome of a species i) First dataset is only V4 region (using #515f- TGCCAGCMGCCGCGGTAA and #806r GGACTACNNGGGTATCTAAT primer) ii). 2nd dataset is V3-V4 region (using standard Illumina primers Forward PrimerTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and Reverse Primer = TCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) I want to classify only the V4 region from both data sets, Would it be possible to extract the V4 r…

Somehow using cutadapt here would only help to remove the primers and not the sequence of interest, or am I understanding it wrong? I was planning to use the function qiime feature-classifier extract-reads to perform what I want but, also unsure if I am in the right direction. If someome could help, I would be very appreciated!

Thanks in advance!

[colinvwood (Colin Wood)]

Hello @asbarros,

Using cutadapt as suggested in that post will remove the 3' primer and everything downstream of it and the 5' primer and everything upstream of it, giving you only the region of interest.

The qiime feature-classifier extract-reads won't take raw reads (fastq files), which I'm assuming you have--cutadapt will.

[asbarros (André Barros)]

Thanks @colinvwood !

I was really not understanding the function cutadapt... So I only need to give him the primer section from V4 and it's settled then, right?

At what stage should I perform this? I would expect before DADA2, since this trimming will definitively impact on the ASV determination.

[colinvwood (Colin Wood)]

Hello @asbarros,

Correct, pass in the V4 primers that you want to use to trim-paired after demuxing but before dada2. (I'm assuming you have paired reads, if you have single-end reads just use trim-single.)