Library prep protocol for 16s rRNA sequencing by MiSeq

[Genetics Kitty]

Hello everyone,

I'm a PhD student in a lab that just started up and we want to do a microbiome analysis on the gut bacteria in a local beetle species. We plan to use primers designed for the V4/V5 hypervariable region of 16s rRNA and will do paired end (2x250bp) sequencing by Illumina MiSeq. However, my lab has never done this before so we don't have a protocol for the library prep.

I thought I would post here and ask if anyone can provide (or direct me to) an optimized library prep protocol for 16s amplicon sequencing by Illumina MiSeq. I've already looked at the Caporaso 2011 paper as well as the Illumina appnote for 16s MiSeq. I fully understand the process, I just need the step by step instructions of how to get from PCR to sequencing.

Thank you so much for any help you can provide!

[cliffbeall]

There is an article from Patrick Schloss' group that just came out:

http://aem.asm.org/content/79/17/511...c-4a15ad282cf2

[DRYTCYV]

Take a look at this new 16S Illumina protocol

http://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.ilmn

I already test it, and it works!