Combining libraries onto MiSeq

Theory behind mothur
1

Hi Mothur Community,

I am hoping to pool a 16s V4 amplicon library (Schloss/Kozich method) of ~300 samples with an ITS2 amplicon library (~20 samples). Does anyone know if this would be possible? The ITS2 amplicon library is constructed similar to 16s (using the same adapter, barcode, pad… etc). Can the V4 and ITS sequencing primers be spiked into their respective MiSeq cartridge wells (12, 13 and 14) together? Or will there be a concern for primer interactions.

V4Read.1. TATGGTAATTGTGTGCCAGCMGCCGCGGTAA
V4Read.2. AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT
V4Index. ATTAGAWACCCBDGTAGTCCGGCTGACTGACT

ITSRead.1. TATGGTAATTGGTCCTCCGCTTATTGATATGC
ITSRead.2. AGTCAGTCAGCCGTGARTCATCGAATCTTTG
ITSIndex (RC of Read.2) CAAAGATTCGATGARTCACGGCTGACTGACT

2

That will/should work. I’ve been doing the same for v4 + other custom primers. I’m adding 6ul 100mM of each v4 primer and 3ul 100mM of each of the other primers. I started doubling the v4 primers a few months ago after having r2 failures that Illumina blamed on the 62-66C annealing temp of the v4 primers-not proof but I haven’t seen the same r2 failures since starting that. I asked Illumina how many primers I could spike in before starting to run into concentration issues and they didn’t seem to think that there would be any issue using 5 or even 10 different custom primers (I’ve done as many as 3)