TITLE: Modified EMP ITS Illumina Amplicon Protocol 
AUTHORS: Dylan P. Smith, Kabir G. Peay, Gail Ackermann, Amy Apprill, Markus Bauer, Donna Berg-Lyons, Jason Betley, T. D. Bruns, J. Greg Caporaso, Noah Fierer, Louise Fraser, Jed A. Fuhrman, M. Gardes, Jack A. Gilbert, Niall Gormley, Greg Humphrey, James Huntley, Janet K. Jansson, Rob Knight, Chris L. Lauber, S. Lee, Sarah M. Owens, Alma E. Parada, Geoff Smith, J. Taylor, Luke Thompson, Willam A. Walters, T. J. White

[DESCRIPTION]
The ITS protocol detailed here is designed to amplify fungal microbial eukaryotic lineages using paired-end community sequencing on the Illumina platform with primers ITS1f-ITS2 (EMP.ITSkabir).

Note: This a modified version used in biocrust project at University of California Riverside.

[BEFORE_START]
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]
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. Primer constructs were designed by Dylan Smith and Kabir Peay.
 
Illumina ITS Primer Constructs (ITS1f-ITS2)
 
Note: Unlike the 16S and 18S sequencing primers, the ITS sequencing primers have additional 3′ bases beyond the PCR primers, in order to match the melting temperature of the Illumina adapters. The forward sequencing primer has 19 and the reverse sequencing primer has 15 additional 3′ bases; therefore the amplicon sequences will begin 19 bp (forward read) and 15 bp (reverse read) after the PCR primers. 
 
EMP.ITSkabir forward primer (ITS1f)
Field descriptions (space-delimited):
5′ Illumina adapter
Forward primer linker
Forward primer (ITS1f; Note: This is 38 bp upstream of ITS1 from White et al., 1990.)
 

AATGATACGGCGACCACCGAGATCTACAC GG CTTGGTCATTTAGAGGAAGTAA


 


 


 

 
EMP.ITSkabir reverse primer (ITS2), barcoded
Field descriptions (space-delimited):
Reverse complement of 3′ Illumina adapter
Golay barcode
Reverse primer linker
Reverse primer (ITS2; Note: This is identical to ITS2 from White et al., 1990.)
 

CAAGCAGAAGACGGCATACGAGAT NNNNNNNNNN CG GCTGCGTTCTTCATCGATGC


 


 


 


 


 


 


 


 


 

 
 
 PCR reaction mixtures
 

 


 

 
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 
ITS amplification 
Primers: ITS1f-ITS2
Amplicon size: ~230 bp
 

 

 
References
   Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N., Owens, S. M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J. A., Smith, G., & Knight, R. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621–1624. http://doi.org/10.1038/ismej.2012.8
 Gardes, M., & Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts. Molecular Ecology, 2(2), 113–118. http://doi.org/10.1111/j.1365-294X.1993.tb00005.x
 Smith, D. P., & Peay, K. G. (2014). Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequencing. PLoS ONE, 9(2), e90234–e90234. http://doi.org/10.1371/journal.pone.0090234
 Walters, W., Hyde, E. R., Berg-Lyons, D., Ackermann, G., Humphrey, G., Parada, A., Gilbert, J. A., Jansson, J. K., Caporaso, J. G., Fuhrman, J. A., Apprill, A., & Knight, R. (2016). Improved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. mSystems, 1(1), e00009–15. http://doi.org/10.1128/mSystems.00009-15
 White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols: a guide to methods and applications (pp. 315–322). New York: Academic Press.
   
 
  
 
 

[STEPS]
SECTION: Amplification Protocol
1. Amplify samples in triplicate. 
 
SECTION: Amplification Protocol
2. Pool triplicate PCR reactions for each sample into a single volume (75 µL). Do not combine amplicons from different samples at this point.
SECTION: Amplification Protocol
3. Run amplicons from each sample on an agarose gel. 
 
 
SECTION: Amplification Protocol
6. 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.
 
SECTION: Amplification Protocol
7. Measure concentration and A260/A280 ratio of final pool that has been cleaned. 


 
SECTION: Amplification Protocol
8. Send an aliquot for sequencing along with sequencing primers listed below.
 
ITS sequencing primers
Read 1 sequencing primer
Field descriptions (space-delimited):
Forward primer segment
Extended region into amplicon
 

TTGGTCATTTAGAGGAAGTAA AAGTCGTAACAAGGTTTCC


a

 
Read 2 sequencing primer
Field descriptions (space-delimited):
Reverse primer segment
Extended region into amplicon
 

CGTTCTTCATCGATGC VAGARCCAAGAGATC


a

 
Index sequencing primer
Reverse complement of extended amplicon region
Reverse complement of reverse primer
Reverse complement of linker
 

TCTC GCATCGATGAAGAACGCAGC CG

SECTION: Amplification Protocol
4. Quantify amplicons (DNA concentration) with NanoDrop spectrophotometer.


SECTION: Amplification Protocol
5. Clean up the amplicons using magnetic bead clean up.  Please contact Matthew Collin matthew.collin@ucr.edu for the bead clean up protocol.