[Vinz]

Our instrument was upgraded a month ago and I just did our first 16S amplicon sequence run and the data was fine (greater than 90% over Q30).

You must be overloading. We run 5pM and 30% PhiX.

That sounds very promising. What setup do you use? How many different primers? Was this a 2x250bp run? Could you post a picture of your %base graph?

[NextGenSeq]

5.5 million reads passing filter. I'll try to post a pic.

[costamc]

Hi,
I am new using Illumina MiSeq. Does anybody have any suggestion on which region of the 16S rRNA gene to use for the new chemistry that amplifies 2x250bp? I was hoping to get an overlap of around 50bp.
Thanks in advance.

[bstamps]

Just replied to this in http://seqanswers.com/forums/showthread.php?t=16812

[costamc]

Hi vs92,

We've been playing around with both the 16S V4 protocol by Caporaso et al. as well as an expanded V4/5 version using a pseudo 2x250 setup for a few months now.

mcnelson.phd,

Which set of primers are using for this region and what is the amplicon size? How many bp overlap when you use the 2X250 setup?

Many thanks.

[mcnelson.phd]

I'm in the process of writing up a manuscript on our use of the v4v5 primers so I can't say a lot about it right now, but we used established primers for 454 amplicons and retro-fit them into the Illumina format. The full size of the amplicon is ~535bp with the adapters included, and the overlap that we get is ~100 bp.

Sorry that I can't really comment more on them.

[CarlHSLee]

vs92 - e-mail if you have time

Hi vs92, please e-mail me at [email protected]

I have a solution to your MiSeq question.

Thanks

[fozrun]

I'm in the process of writing up a manuscript on our use of the v4v5 primers so I can't say a lot about it right now, but we used established primers for 454 amplicons and retro-fit them into the Illumina format. The full size of the amplicon is ~535bp with the adapters included, and the overlap that we get is ~100 bp.

Sorry that I can't really comment more on them.

Hi:

Is your manuscript closer to publication? I'm looking at getting some primers made up for diversity studies for a new MiSeq that will be arriving soon. I'd rather not start from scratch. Did you use the staggered bases to avoid using PhiX?

Any help appreciated. you can e-mail forsterr at agr dot gc dot ca

[costamc]

Hi again,
We've decided to go with the V3-V4 region and was hoping to use my forward and reverse primers as my sequencing primers and the reverse compliment of the reverse primer as my index primer. The problem I just found during a real time PCR to determine sequencing primer efficiency is that the primers will not bind their targets efficiently at the required temperature (65oC).
We can easily increase the length of the sequencing primers going back into the Illumina adapters, but I am afraid that when I increase the length of the index primer I will end up out of the conservative region. I've tried including several degeneracies, but the lowest Tm is never higher than 65oC.
the reverse primer I choose is the S-D-Bact-0785-a-A-21: 5′-GACTACHVGGGTATCTAATCC-3

I appreciate any ideas or comments.

Marcio.

[JamieHeather]

You could try using modified oligos, such as by introducing LNA bases, which should make it anneal at a lower temperature.

[tallcuppajoe]

Hello Marcio. We are also using that reverse primer but are running into some issues with concatamers or primer dimers. Are you experiencing similar issues?

Joe

[costamc]

Hi Joe,
I haven't gotten any results yet because we are having some technical issues with our MiSeq, so I can not comment on your question yet. However, I had a lot problems with short reads in the past when I was using 454 to sequence 16S amplicons that had the same forward primer. We even had to change our purification protocol as recommended by the technical bulletim 2011-007 (January 2012), but I ended up skipping the step in which you replace the AMPure buffer by sizing solution (or it would yield no DNA after purification).
Marcio.

[victorsor]

We have performed an experiment in which have obtained about 5KK reads PF of 16S.
Strategy:
Caporaso primers with CS1-CS2 tails for a second PCR for indexing
Loaded at 10 pM with 50% Phix
Sequencing (2x151) with a mixed custom-Truseq primers for read1 and read2
CS1 reverse primer for index read
RESULTS:
About 11M raw clusters, more than 87% PF, more than 87% >Q30
51% of clusters PF correspond to 16S.
Very, very good quality of read1, slightly badder quality in read 2 mainly after about 120 cycle.

[urchin]

I'm in the process of writing up a manuscript on our use of the v4v5 primers so I can't say a lot about it right now, but we used established primers for 454 amplicons and retro-fit them into the Illumina format. The full size of the amplicon is ~535bp with the adapters included, and the overlap that we get is ~100 bp.

Sorry that I can't really comment more on them.

I wanted to ask McNelson.Phd if he has now published this work. If so, please give a link to the paper. I am trying to work out a project using 16s rRNA gene now and need to keep the costs low. We are just starting and have very little help from anyone. The slightly more experienced lab that was advising us just found out their materials were not adequate for use with Miseq when they submitted them.