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SUPPLEMENTARY INFORMATION
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Supplementary Methods
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Classification of ANAS contigs by tetranucleotide frequencies. ANAS contigs larger than
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2,500 bp were grouped by tetranucleotide frequencies (TF) using a procedure based on one
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described by Dick et al. (Dick et al 2009) with some modifications described here. Clustered
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regularly interspaced short palindromic repeat (CRISPR) and rRNA gene sequences were
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removed from contig sequences prior to classification because these sequence regions are known
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to have atypical nucleotide compositions compared to their genomes (Dick et al 2009, Reva and
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Tummler 2005). Next, all contig sequences larger than 2500 bp were selected for classification,
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with contigs larger than 7500 bp divided into 5000 bp fragments. Sequence fragments were
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classified based on TF using the Databionics ESOM Tools program (Databionics 2006, Ultsch
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and Moerchen 2005). Dick et al.’s method for clustering of sequences (Dick et al 2009) was
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used with the following modifications. Online training was used instead of the k-batch algorithm
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because online training provides more accurate, albeit slower, performance (Databionics 2006).
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A map size of 120x196 and an initial radius of 60 were selected based on the size of the dataset.
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PhyloChip Assessment of Community Composition. Total nucleic acids were extracted as
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previously described (West et al 2008). DNA and RNA were separated using the Qiagen
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AllPrep DNA/RNA Kit according to manufacturer’s instructions. RNA was further purified
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using the Qiagen RNase-free DNase Set, per manufacturer’s instructions. RT-qPCR was
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performed to confirm that RNA samples contained no DNA contamination. The masses of DNA
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and RNA per volume were quantified using a fluorometer [model TD-700, Turner Designs,
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Sunnyvale, CA] and the Quant-iT PicoGreen dsDNA and Quant-iT RiboGreen RNA reagents
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[Invitrogen Molecular Probes, Carlsbad, CA], respectively, according to the manufacturer's
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instructions.
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The bacterial and archaeal 16S rRNA genes were amplified from the extracts using the
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following primers: bacterial primer 27F (5′-AGRGTTTGATCMTGGCTCAG), archaeal primer
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4F (5'-TCC GGT TGA TCC TGC CGG-3'), and universal primer 1492R (5′-
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GGTTACCTTGTTACGACTT). For DNA PhyloChips, PCR was performed using the TaKaRa
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Ex Taq system [Takara Bio Inc., Japan] and DNA was prepared for the microarrays as previously
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described (DeSantis et al 2007).
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For RNA PhyloChips, a direct hybridization method was employed as follows. 16S rRNA
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was enriched from total RNA by gel extraction. Direct analysis of rRNA was achieved using a
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modification of the protocol of Cole et al.(Cole et al 2004). To account for technical variation
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between hybridizations, a set of internal RNA spikes were added to each sample preparation.
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These spikes consisted of transcripts generated by T7 or T3 mediated in vitro transcription from
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linearized plasmids pGIBS-LYS (containing Bacillus subtilis lysA, ATCC 87482), pGIBS-PHE
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(containing Bacillus subtilis Phe gene, ATCC 87483) and pGIBS-THR (containing Bacillus
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subtilis Thr gene, ATCC 87484). To each RNA fragmentation reaction, 1.35×1010, 3.13×1010
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and 3.13×1011 transcripts of LysA, Thr, and Phe respectively were added in a volume of 1 μL.
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Combined sample RNA (1 µg) and spike mix was fragmented and dephosphorylated
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simultaneously using 0.1U RNaseIII/µg RNA, shrimp alkaline phosphatase [USB, OH, USA]
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0.2U/µg RNA in a buffer containing 10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1mM DTT
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(pH7.9) in a final volume of 20 µL. The mixtures were then incubated at 37°C for 35 min
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followed by inactivation at 65°C for 20 min. RNA labeling with multiple biotin residues utilized
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an efficient labeling system that employs T4 RNA ligase to attach a 3'-biotinylated donor
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molecule [pCp-Biotin3, Trilink Biotech, San Diego, CA, USA] to target RNA (Cole et al 2004).
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Labeling was performed with 20 µL of fragmented/dephosphorylated RNA, 20U T4 RNA ligase
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[NEB, MA, USA], 100 µM pCp-Biotin3 in a buffer containing 50 mM Tris-HCl, 10 mM MgCl2,
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10 mM DTT, 1 mM ATP (pH 7.8), 16% v/v PEG 8000. The final volume was 45 µL. The
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reaction mixture was incubated at 37°C for 2h and inactivated at 65°C for 15 min. The mixture
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was then prepared for PhyloChip hybridization without any further purification and was
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processed according to standard Affymetrix expression analysis technical manual procedures for
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cDNA.
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PhyloChips were hybridized at 50°C in an Affymetrix hybridization oven for 16 h at 60 rpm.
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Microarrays were stained according to the Affymetrix protocol and then immediately scanned
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using a GeneChip Scanner 3000 7G [Affymetrix, Santa Clara CA]. To process captured
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fluorescent images into taxon hybe scores, images were background corrected and probe pairs
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scored as previously described (Brodie et al 2006).
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REFERENCES
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Brodie EL, DeSantis TZ, Joyner DC, Baek SM, Larsen JT, Andersen GL et al. (2006).
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Application of a high-density oligonucleotide microarray approach to study bacterial
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population dynamics during uranium reduction and reoxidation. Appl Environ Microbiol 72:
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6288-6298.
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Cole K, Truong V, Barone D, McGall G. (2004). Direct labeling of RNA with multiple biotins
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allows sensitive expression profiling of acute leukemia class predictor genes. Nucleic Acids
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Res 32: e86.
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Databionics (2006). Databionic ESOM Tools User Manual.
[http://databionic-esom.sourceforge.net/].
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DeSantis TZ, Brodie EL, Moberg JP, Zubieta IX, Piceno YM, Andersen GL. (2007). High-
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density universal 16S rRNA microarray analysis reveals broader diversity than typical clone
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library when sampling the environment. Microb Ecol 53: 371-383.
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Dick GJ, Andersson AF, Baker BJ, Simmons SL, Thomas BC, Yelton AP et al. (2009).
Community-wide analysis of microbial genome sequence signatures. Genome Biol 10: R85.
Reva ON, Tümmler B. (2005). Differentiation of regions with atypical oligonucleotide
composition in bacterial genomes. BMC Bioinformatics 6: 251.
Ultsch A, Mörchen F. (2005). ESOM-Maps: tools for clustering, visualization, and classification
with Emergent SOM. Technical Report, University of Marburg, Germany.
West KA, Johnson DR, Hu P, DeSantis TZ, Brodie EL, Lee PKH et al. (2008). Comparative
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genomics of "Dehalococcoides ethenogenes" 195 and an enrichment culture containing
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unsequenced "Dehalococcoides" strains. Appl Environ Microbiol 74: 3533-3540.