EMP 18S Illumina Amplicon Protocol

Markus Bauer, Donna Berg-Lyons, Jason Betley, J. Greg Caporaso, Noah Fierer, Louise Fraser, Jack A. Gilbert, Niall Gormley, James Huntley, Janet K. Jansson, Rob Knight, Chris L. Lauber, Sarah M. Owens, Geoff Smith, Luke Thompson, William A. Walters, Linda A. Amaral-Zettler, Hugh W. Ducklow, Susan M. Huse, Simon N. Jarman, Elizabeth A. McCliment, Hege Vestheim

Published: 2023-02-17 DOI: 10.17504/protocols.io.ewov1b6pgr24/v2

Abstract

The 18S protocol detailed here is designed to amplify eukaryotes broadly with a focus on microbial eukaryotic lineages. The primers target the 18S SSU rRNA and are based on those of Amaral-Zettler et al. (2009). The constructs are designed to be used with the Illumina platform.

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.

The outlines of the protocol are the same as the 16S protocol, but different primers, PCR conditions, and sequencing primers are used. In addition, we have designed a blocking primer that reduces the amplification of vertebrate host DNA to be used on host-associated samples, especially those that have a low eukaryotic biomass. Blocking primer strategy is based on Vestheim et al. (2008).

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).

Note

Do not combine amplicons from different samples at this point.

Run amplicons from each sample on an agarose gel.

Note

Expected band size for 1391f-Eukbr is ~260 bp. Low-biomass samples may yield faint or no visible bands; alternative methods such as a Bioanalyzer could be used to verify presence of PCR product.

Quantify amplicons with Quant-iT PicoGreen dsDNA Assay Kit (follow manufacturer’s instructions).

Quanti-iT™ Pico Green dsDNA Assay (Invitrogen P7589)

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.

Note

When working with multiple plates of samples, it is typical to produce a single tube of amplicons for each plate of samples.

Clean amplicon pool using MoBio UltraClean PCR Clean-Up Kit (follow manufacturer’s instructions).

Note

If working with more than 96 samples, the pool may need to be split evenly for cleaning and then recombined.Optional : If spurious bands were present on gel (in step 3), one-half of the final pool can be run on a gel and then gel extracted to select only the target bands.

Measure concentration and A260/A280 ratio of final pool that has been cleaned.

Note

For best results the A260/A280 ratio should be between 1.8-2.0.

Send an aliquot for sequencing along with sequencing primers listed in Guidelines.

EMP 18S Illumina Amplicon Protocol

Abstract

Before start

Steps

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