MiSeq v3 600 cycle for amplicons

Hi all

This isn’t a mothur specific question but a general survey of amplicon sequence on MiSeq type people.

How many have tried the newer 600 cycle kits? We’re seeing substantial quality decreases in both directions (<q30 by ~200 in the forward read and ~450 (base 150) reverse. <q20 ~240 forward and 510 (base 210)reverse). This is our first test of the 600 cycle kit, we were clustering at 1250 and it was a genome run not even amplicons. Compare those numbers to the quality that we see in a 250cycle v2 amplicon run that’s clustered at 600 (<q30 on cycle 250 and 450, <q20 on 500)

Anyone else seeing this? I’m having people want to use MiSeq for other amplicons that are 400-600bp, who believe that a v3 kit will give them 300 good bases in both directions or at the minimum 250 good bases. I’m not seeing that as a possibility.



Not sure if you’ve seen this…


We do see the problems with the v3 chemistry. I’m also curious whether people see quality plots like the one there. We’ve repeated this numerous times with different loading densities, presented these data at an Illumina Users Meeting, and Illumina is like, “So what. What’s your question?”

Also, I’ve heard from other people at UMich that since the beginning of the year v2 kits have been having problems too.

Hi Pat

I didn’t see that till after I posted the question, so thanks for reposting.

I’m now now sure if the horrible quality is just the v3 or if there is something off with our MiSeq. The run right after the horrible but barely in specs v3 run completely failed (60% >q30) so a tech is coming out to check the optics.

I’ve only been in charge of this MiSeq for a few months so haven’t gone to a user meeting yet, but get a very strong impression from tech support that they have little experience (or interest?) in tuning it for amplicons. I’ll try to grab the quality plot and post it here, but your’s looks way, way better than our run

Illumina has finally officially said that they’re having issues with the 500 and 600 cycle kits. They don’t know what the issue is, so right now there’s no telling if the replacement kit will have the same quality fall off. But at least they’re finally acknowledging the problem

FWIW, we’ve been having V3 problems since they released the kit.