Primer orientations in illumina miseq metabarcoding data

[SoilRotifer (Mike Robeson)]

I think these threads may help:

How to trim primers from mixed oriented reads:

q2-cutadapt output

Good news @jessica.song! I think I solved this. First, thank you for sending me the PDF describing the protocol. I noticed a key statement: pairs of primers (Fw-Rev or Rev-Fw) had to be present in the sequence fragments That is, this particular sequencing facility must expect mixed orientation reads. Therefore you need to enter in both primers for each of the --p-front-* commands (in 5' - 3' orientation). Like so: qiime cutadapt trim-paired \ --i-demu…

Potential way to orient fastqs after trimming primers:

Preparing mixed orientation reads for dada2

Hi @shreyaramachandran, This may help.. though a bit onerous. That being said... We've been discussing ways to implement the vsearch --orient ... command into QIIME 2 for orienting fastq files. You could try running this vsearch command externally on each of your R1 and R2 reads, or you can use reads that are already merged. vsearch is available within your QIIME 2 environment. It might also be easiest to run this prior to importing into QIIME 2. tl;dr: vsearch \ --orient R1-seqs.fastq…