TITLE: EMP 16S Illumina Amplicon Protocol
AUTHORS: J. Greg Caporaso, Gail Ackermann, Amy Apprill, Markus Bauer, Donna Berg-Lyons, Jason Betley, Noah Fierer, Louise Fraser, Jed A. Fuhrman, Jack A. Gilbert, Niall Gormley, Greg Humphrey, James Huntley, Janet K. Jansson, Rob Knight, Chris L. Lauber, Catherine A. Lozupone, Sean McNally, David M. Needham, Sarah M. Owens, Alma E. Parada, Rachel Parsons, Geoff Smith, Luke R. Thompson, Luke Thompson, Peter J. Turnbaugh, William A. Walters, Laura Weber

[DESCRIPTION]
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.

[GUIDELINES]
16S amplification primers 
 
The current primers have been modified from the original 515F-806R primer pair (Caporaso et al., 2011) in the following ways:
 
Barcodes are now on the forward primer of the new 515FB-806RB primer pair. This enables the usage of various reverse primer constructs to obtain longer amplicons (tested on 806RB and 926R).
Degeneracy was added to both the forward and reverse primers to remove known biases against Crenarachaeota/Thaumarchaeota (515FB, also called 515F-Y, Parada et al., 2016) and the marine and freshwater Alphaproteobacterial clade SAR11 (806RB, Apprill et al., 2015).
The primer sequences without linker, pad, barcode, or adapter are as follows:
 
Current, 2015-present (fwd-barcoded: 515FB-806RB): 

FWD:GTGYCAGCMGCCGCGGTAA; REV:GGACTACNVGGGTWTCTAAT

Original, pre-2015 (rev-barcoded: 515F-806R): 

FWD:GTGCCAGCMGCCGCGGTAA; REV:GGACTACHVGGGTWTCTAAT

 
Note: Studies in the Qiita database will have 
library_construction_protocol
 
as 
515f/806rbc
 if original primers or 
515fbc/806r
 if current primers (“bc” stands for barcode).
 
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 for more information. Primers and primer constructs were designed by Greg Caporaso (2011, 2012). Modifications to primer degeneracy were done by the labs of Jed Furhman (Parada et al., 2016) and Amy Apprill (Apprill et al., 2015). Forward-barcoded constructs were redesigned by Walters et al. (2016) based upon the original constructs generated by Caporaso et al. (2012).
 
Illumina 16S Primer Constructs (shorter: 515FB–806RB)
Illumina 16S Primer Constructs (longer: 515FB–926R)
 
515FB forward primer, barcoded:
Field descriptions (space-delimited):
5′ Illumina adapter
Golay barcode
Forward primer pad
Forward primer linker
Forward primer (515FB)

AATGATACGGCGACCACCGAGATCTACACGCT XXXXXXXXXXXX TATGGTAATT GT GTGYCAGCMGCCGCGGTAA

 
806RB reverse primer:
Field descriptions (space-delimited):
Reverse complement of 3′ Illumina adapter
Reverse primer pad
Reverse primer linker
Reverse primer (806RB)

CAAGCAGAAGACGGCATACGAGAT AGTCAGCCAG CC GGACTACNVGGGTWTCTAAT

 
 
PCR reaction mixture 
 

 

 
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
 
Thermocycler conditions 
Primers: 16S V4 515f-806rB
Amplicon size: ~390 bp
Cycle times are longer for 384-well thermocyclers.
 

 



16S sequencing primers 
 
Read 1 sequencing primer: 
Field descriptions (space-delimited):
Forward primer pad
Forward primer linker
Forward primer

TATGGTAATT GT GTGYCAGCMGCCGCGGTAA

 
Read 2 sequencing primer: 
Field descriptions (space-delimited):
Reverse primer pad
Reverse primer linker
Reverse primer

AGTCAGCCAG CC GGACTACNVGGGTWTCTAAT


Index sequencing primer: 
Note: The 5′ adapter sequence/index sequencing primer has an extra GCT at its 3′ end compared to Illumina’s usual index primer sequences. These bases were added to the 3′ end of the Illumina 5′ adapter sequence to increase the Tm for read 1 during sequencing.

AGTCAGCCAG CC GGACTACNVGGGTWTCTAAT

 
References 
Apprill, A., McNally, S., Parsons, R. & Weber, L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat Microb Ecol 75, 129–137 (2015). doi:10.3354/ame01753
Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci USA 108, 4516–4522 (2011). doi:10.1073/pnas.1000080107
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
Parada, A. E., Needham, D. M. & Fuhrman, J. A. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ Microbiol 18, 1403–1414 (2016). doi:10.1111/1462-2920.13023
Walters, W. et al. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys. mSystems 1, 915 (2016). doi:10.1128/mSystems.00009-15

[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.