EMP 16S Illumina Amplicon Protocol
Gail Ackermann, Amy Apprill, Markus Bauer, Donna Berg-Lyons, Jason Betley, J. Greg Caporaso, Noah Fierer, Louise Fraser, Jed A. Fuhrman, Jack A. Gilbert, Niall Gormley, Greg Humphrey, James Huntley, Janet K. Jansson, Rob Knight, Chris L. Lauber, Sarah M. Owens, Alma E. Parada, Geoff Smith, Luke Thompson, Catherine A. Lozupone, Sean McNally, David M. Needham, Rachel Parsons, Luke R. Thompson, Peter J. Turnbaugh, William A. Walters, Laura Weber
Abstract
The 16S protocol detailed here is designed to amplify prokaryotes (bacteria and archaea) using paired-end 16S community sequencing on the Illumina platform. Primers 515F-806R target the V4 region of the 16S SSU rRNA.
For running these libraries on the MiSeq and HiSeq, please make sure you read the supplementary methods of Caporaso et al. (2012). You will need to make your sample more complex by adding 5-10% PhiX to your run.
Before start
Steps
Amplify samples in triplicate, meaning each sample will be amplified in 3 replicate 25-µL PCR reactions.
Pool triplicate PCR reactions for each sample into a single volume (75 µL).
Run amplicons from each sample on an agarose gel.
Quantify amplicons with Quant-iT PicoGreen dsDNA Assay Kit (follow manufacturer’s instructions).
Combine an equal amount of amplicon from each sample (240 ng) into a single, sterile tube. Higher amounts can be used if the final pool will be gel-isolated or when working with low-biomass samples.
Clean amplicon pool using MoBio UltraClean PCR Clean-Up Kit (follow manufacturer’s instructions).
Measure concentration and A260/A280 ratio of final pool that has been cleaned.
Send an aliquot for sequencing along with sequencing primers listed in Guidelines.
