Workflow for illumina demultiplexed paired end data

[Dharitri (dharitri saikia)]

Dear community,
I have miseq paired end illumina (2*250) data for 5 samples (10 seperate fastq files, each with R1 and R2). samples contain the forward and reverse primers but not the barcodes. till now, I’ve removed the primers and adapters, checked the quality, joined the forward and reverse reads. but after that I’m stuck. For otu picking and biom file generation, should i go for each sample separately or I have to merge all the joined (R1 and R2 for each, fasta) 5 seperate files for 5 samples together in a single fasta file with proper label and then go for otu picking and other downstream analysis? I appreciate your help in advance.

[Nicholas_Bokulich]

Hi @Dharitri,
Import the sequence files together into a single artifact of demultiplexed sequences, and go from there.

Import as described here or here. Either will produce a single artifact of demultiplexed sequences that you can use downstream.

Then you can proceed with qiime dada2 denoise-paired or with OTU picking (if you use dada2 you can trim the primers out with the trim parameters. If you use OTU picking, first use cutadapt trim-paired as described here to trim primers before proceeding).

Good luck!

[Dharitri (dharitri saikia)]

Dear Nicholas,
I have followed as you suggested up to core-metrics-phylogenetics. all the output files have been generated. but now when I'm trying to run qiime diversity alpha-group-significance i am repeatedly getting error msg as uploaded here. Kindly help me.

[Mehrbod_Estaki]

Hi @Dharitri,

This error message indicates a regrouping issue in your metadata file in that there are no ‘grouping’ terms for the script to use. See this error and solutions in other posts here and here for example.