Supplementary Methods
16S rDNA 454-Pyrosequencing and data analyses.
DNA extracts were normalized to 10 ng/l prior to PCR. Samples were amplified
using a universal primer pair targeting the 16S rDNA V9 region. The forward primer was
515F (‘5-GTGCCAGCMGCCGCGGTAA-‘3) and the reverse primer was 907R (‘5CCGTCAATTCCTTTRAGTTT-‘3). The forward and reverse primers were downstream
of the FLX-454 primer adapters and the reverse primer also contained a 12-bp barcode
unique to a specific sample (Hamady et al. 2008). PCR reactions were performed using
Takara Ex Taq polymerase (Takara, Madison, WI), with the following thermocycling
parameters: initial denaturation at 95 C for 1 minute followed by 25 cycles of 95 C for
20 seconds, 30 seconds of annealing at 66 C and extension at 72 C for 1 minute. Final
product extension was at 72 C for 10 minutes. Reaction primer dimmers were removed
from the PCR products via SPRI bead purification according to the manufacturer’s
protocol (AMPure XP, Beckman Coulter Genomics, Danvers, MA) before being checked
for quality and quantity on a Bioanalyzer 2100 using a DNA 7500 chip (Agilent
Technologies, Santa Clara, CA). Each PCR sample was normalized to 30 ng and pooled
together for multiplex sequencing. Sequencing libraries were created using the SV emuPCR kit (Lib-A, Roche, Indianapolis, IN) and sequencing on a GS-FLX sequencer
(Roche, Indianapolis, IN) at the Veterans Medical Research Foundation, La Jolla, CA.
Reads between 200 and 1000 bp were used for downstream analysis in the
Quantitative Insights Into Microbial Ecology (QIIME) pipeline (average = 426 bp)
(Caporaso et al. 2010a. All sequences with a mean quality score below 25 and
homopolymer run of 6 or more were removed. The total number of reads amounted to
56,182 reads with an average number of 6,208 sequences per sample (range: 1,90014,595). All reads were deposited at the European Bioinformatics Institute (EBI) with the
accession number ERP003515. Sequences were clustered at 97% pairwise identity using
the UCLUST (Edgar, 2010) reference-based OTU picking method, where the reference
dataset was the GreenGenes 99% (GG99_12_10, Werner et al. 2012) data set. A
representative sequence from each OTU was aligned to the GG99 data set using PyNAST
(Caporaso et al., 2010b). The concatenated alignment of OTUs was filtered to remove
gaps and hypervariable regions using the GreenGenes Lane mask (DeSantis et al. 2006).
A phylogenetic tree was constructed from the filtered alignment using FastTree (Price et
al., 2010). Taxonomic assignments were done with the naïve Bayesian algorithm (Wang
et al., 2007) developed for the RDP classifier (Cole et al., 2009) using the GG99
taxonomy data set as training (Werner et al. 2012). Phylogenetic -diversity was
quantified with a weighted Unifrac distance matrix which was constructed at a depth of
coverage of 19,000 sequence (normalized coverage to the minimum number of sequences
in the dataset). The resulting distance matrix was used to cluster samples using UPGMA
hierarchical clustering algorithm. Support values for the UPGMA dendrograms were
calculated using 100 jackknife permutations and nodes having at least 50% support were
deemed to have high support.
Supplementary References
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello,
EK, Fierer N, Pena A.G, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D,
Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J,
Sevinsky JR, Tumbaugh PJ, Walters WA, Wildmann J, Yatsunenko T, Zaneveld J,
Knight R. (2010a). QIIME allows analysis of high-throughput community sequencing
data. Nat Methods 7: 335-336.
Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R,
(2010b). PyNAST: a flexible tool for aligning sequences to a template alignment.
Bioinformatics 26: 266-267.
Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen
AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM. (2009). The Ribosomal Database
Project: improved alignments and new tools for rRNA analysis. Nucl Acids Res 37:
D141-D145.
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T,
Dalevi D, Hu P, Andersen GL. (2006). Greengenes, a chimera-checked 16S rRNA gene
database and workbench compatible with ARB. Appl Environ Microbiol 72: 5069-5072.
Edgar RC. (2010). Search and clustering orders of magnitude faster than BLAST.
Bioinformatics 26:2460-2461.
Hamady M, Walker JJ, Harris JK, Gold NJ, Knight R. (2008). Error-correcting
barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat Methods 5:
235-237.
Price MN, Dehal PS, Arkin AP. (2010). FastTree2- approximately maximumlikelihood trees for large alignments. PloS One 5: e9490.
Reeder J, Knight R. (2010). Rapidly denoising pyrosequencing amplicon reads by
exploiting rank-abundance distributions. Nat Methods 7: 668-669.
Werner JJ, Koren O, Hugenholtz P, DeSantis TZ, Walters WA, Caporasso JG,
Angenent LT, Knight R, Ley RW. (2012). Impact of training sets on classifications of
high-throughput bacterial 16S rRNA gene surveys. ISME J 6:94-103.