Determining correct pre-processing pipeline for

[thermokarst]

Hey there @Kara!

@Kara

We are working through our first trial microbiome study after reading tutorials and forums for weeks!

@Kara

A) import paired-end reads to create QIIME artifact
*B) join paired-end reads (do we need to worry about correctly orienting the R1 and R2 reads?)
C) demultiplex data and cut barcodes/primers with “demux-paired” and “trim-paired” commands which use cutadapt plugin
E) quality filter and denoise with DADA2

I would skip step B if you are planning on using DADA2. More on that shortly...

@Kara

thread that DADA2 will join paired-end reads, so we shouldn’t use joined data as the input for DADA2, is that correct?

That is exactly the case - DADA2 works best with unprocessed data - part of the processing in DADA2 will join the reads for you. In some cases where overlap is insufficient or reverse reads are poor quality, it can be advantageous to run DADA2 on just the forward reads, but we will cross that bridge when we get there.

For now, I would recommend proceeding with A, C, E (btw, where did D go??), with one caveat. Adata-integrity bug was just identified this week with q2-cutadapt's trim-paired method. I would recommend waiting until the 2018.6 release comes out before proceeding (we are on track for releasing later today ).

Keep us posted!