TITLE: EMP 18S Illumina Amplicon Protocol
AUTHORS: Linda A. Amaral-Zettler, Markus Bauer, Donna Berg-Lyons, Jason Betley, J. Greg Caporaso, Hugh W. Ducklow, Noah Fierer, Louise Fraser, Jack A. Gilbert, Niall Gormley, James Huntley, Susan M. Huse, Janet K. Jansson, Simon N. Jarman, Rob Knight, Chris L. Lauber, Elizabeth A. McCliment, Sarah M. Owens, Geoff Smith, Luke Thompson, Hege Vestheim, William A. Walters

[DESCRIPTION]
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).

[GUIDELINES]
Ordering primers 
 
The primer sequences in this protocol are always listed in the 5′ -> 3′ orientation. This is the orientation that should be used for ordering. See the page Primer Ordering and Resuspension. Primer constructs were designed by Laura Wegener Parfrey.
Illumina 18S Primer Constructs (Euk1391f-EukBr)
 
Illumina_Euk_1391f forward primer: 
Field descriptions (space-delimited): 
5′ Illumina adapter
Forward primer pad
Forward primer linker
Forward primer (1391f)

AATGATACGGCGACCACCGAGATCTACAC TATCGCCGTT CG GTACACACCGCCCGTC


 

 
Illumina_EukBr reverse primer, barcoded: 
 

Reverse complement of 3′ Illumina adapter
Golay barcode
Reverse primer pad
Reverse primer linker
Reverse primer (EukBr)

CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXXXX AGTCAGTCAG CA TGATCCTTCTGCAGGTTCACCTAC

 
Mammal_block_I-short_1391f mammal blocking primer:
The mammal blocking primer is to be used when there is a high probability of picking up host genomic DNA. The C3 spacer (/3SpC3/) is a chemical modification that prevents extension during the PCR. Please note that the use of blocking primer reduces the number of host sequences detected but does not completely eliminate them. Thus remaining host sequences should also be filtered out during the analysis phase. We have found blocking primers to be particularly useful for host-associated samples with a low biomass of eukaryotic DNA. Note: Sequence is formatted for ordering from IDT.
 

GCCCGTCGCTACTACCGATTGG/ideoxyI//ideoxyI//ideoxyI//ideoxyI//ideoxyI/TTAGTGAGGCCCT/3SpC3/

 
 
PCR reaction mixture 
 
No blocking primer

 

 
With blocking primer 

 

 
Notes:
PCR-grade water from Sigma (cat. no. W3500) or MoBio (cat. no. 17000-11)
Platinum Hot Start PCR Master Mix (2x) from ThermoFisher (cat. no. 13000014)
Final master mix concentration in 1x reaction: 0.8x
Final primer concentration in 1x reaction: 0.2 µM
Final blocking primer concentration in 1x reaction: 1.6 µM
 
Thermocycler conditions 
 
No blocking primer: 

 

 
With blocking primer: 

 

 
 
18S sequencing primers 
 
Euk_illumina_read1_seq_primer:
Field descriptions (space-delimited):
Forward primer pad
Forward primer linker
Forward primer (1391f)

AGTCAGTCAG CA TGATCCTTCTGCAGGTTCACCTAC

 
Euk_illumina_read2_seq_primer:
Field descriptions (space-delimited):
Reverse primer pad
Reverse primer linker
Reverse primer (EukBr)

GTAGGTGAACCTGCAGAAGGATCA TG CTGACTGACT


Euk_illumina_index_seq_primer:
Field description (space-delimited):
Reverse complement of reverse primer (EukBr)
Reverse complement of reverse primer linker
Reverse complement of reverse primer pad

GTAGGTGAACCTGCAGAAGGATCA TG CTGACTGACT
 
 
References 
Amaral-Zettler, L. A., McCliment, E. A., Ducklow, H. W. & Huse, S. M. A method for studying protistan diversity using massively parallel sequencing of V9 hyper variable regions of small-subunit ribosomal RNA Genes. PLoS ONE 4, e6372 (2009).
Caporaso, J. G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J (2012). doi:10.1038/ismej.2012.8
Vestheim, H. & Jarman, S. N. Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs. Frontiers in Zoology 2008 5:1 5, 12 (2008).

[STEPS]
1. Amplify samples in triplicate, meaning each sample will be amplified in 3 replicate 25-µL PCR reactions.
2. Pool triplicate PCR reactions for each sample into a single volume (75 µL). 
 
3. Run amplicons from each sample on an agarose gel. 
 
4. Quantify amplicons with Quant-iT PicoGreen dsDNA Assay Kit (follow manufacturer’s instructions).
 
 
5. 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. 
 
6. Clean amplicon pool using MoBio UltraClean PCR Clean-Up Kit (follow manufacturer’s instructions). 
 
 
7. Measure concentration and A260/A280 ratio of final pool that has been cleaned. 
 
8. Send an aliquot for sequencing along with sequencing primers listed in Guidelines.