Gao et al. 10.1073/pnas.0607077104. |
SI Materials and Methods
Specimen Processing. In brief, a 2×2-cm area of the volar (flexor) surface of the
forearm, midway between wrist and elbow was sampled by swabbing the skin with a
cotton pledget that had been soaked in sterile 0.15 M NaCl with 0.1% Tween 20
(Fisher Scientific, Fair Lawn, NJ). DNA was extracted from the swabs in a
PCR-free clean-room by using the DNeasy Tissue Kit (Qiagen, Chatsworth, CA);
because Gram-positive bacteria are more resistant to lysis than Gram-negative
organisms, the manufacturer's protocol for genomic DNA isolation from
Gram-positive bacteria was followed. Samples were eluted in 100 ml of AE
buffer, and to eliminate bacterial or DNA contamination, the enzymatic lysis
buffer was passed through a micro-centrifuge filter (MW threshold 30,000
daltons; Amicon, Bedford, MA) at 747×g for 20 min.
16S rDNA PCR Amplification.
Universal bacterial 16S rDNA PCR primers 8F (forwardprimer 5'-AGA GTT TGA TYM TGG CTC AG) and 1510R (reverse primer 5'-TAC GGY TAC
CTT GTT ACG ACT T) were used to amplify the region corresponding to positions 8
to 1513 of the Escherichia coli 16S rDNA gene by using a 30-cycle PCR. To each 5
ml of the suspension of extracted template DNA was added 45 ml of a PCR
mixture containing 5 ml of 10´ PCR buffer (Qiagen, Valencia, CA), 2.5 mM
MgCl2, 200 mM each dNTP, 20 pmol of each primer, and 5units of TaqDNA
polymerase. PCR was performed for 2 min at 94°C, followed by 30 amplification
cycles of 45 s at 94°C, 30 s at 52°C, and 90 s at 72°C, with a final cycle
for 20 min at 72°C. The results of PCR amplification were examined by
electrophoresis on 1% agarose gels.
16S rDNA Clone Libraries.
Putatively positive clones were screened by PCR with Sp6/T7 primers. The cloned inserts underwent sequence analysis using PCR primers 8F and 27R (reverse primer 5'-CGA CAI CCA TGC AIC ACC T, corresponding to position 8 to 1064 of the E. coli 16S rDNA). Each sequence was manually edited in conjunction with its chromatogram with Sequencher, adjusting for quality. DNA sequences of »980 bases were obtained initially to determine either identity or approximate phylogenetic position. For those clones containing inserts of ambiguous phylogenetic status, nearly full-length 16S bacterial rDNA sequences (»1,400 bp) were obtained, using the additional primer, 1510R.Elimination of Contaminating Sequences.
Because reagents used in DNA extraction and PCRs may contain bacteria or their genomic DNA, and under certain experimental conditions these contaminating DNA molecules may become detectable after PCR amplification, we used a reagent control that included all DNA extraction and PCR reagents but without the skin sample, which was examined in parallel using the identical procedures as for the skin sample DNA. After electrophoresis and ethidium bromide staining, preparations from these controls did not generate any visible bands, but the agarose gel at the expected location of the signal was excised, ligated to pGEM-T Easy Vector (Promega) and transformed. In total, 89 clones derived from three reagent controls underwent sequence analysis (SI Table 2); sequences of 19 known species and 2 unknown species were identified. For a more conservative data analysis, the 10 species found in both control and skin samples were excluded.Sequence Deposition.
All sequences that are not classifiable by using the current 16S database at RDP II were deposited in the GenBank database (accession nos. DQ130020-DQ130049 and DQ847437-DQ847450).Statistical Methods.
Double principal coordinate analysis (DPCoA) uses phylotype differences to derive the dissimilarity matrix of samples and calculate the sample diversity. In this analysis, the dissimilarities between different phylotypes are calculated based on the sum of distance to the common ancestor of two phylotypes on phylotype tree. To facilitate the visualization of sample dissimilarity and diversity, the first two orthogonal principal axes were obtained based on the sample dissimilarity, and were plotted to show the distribution of samples in a two- dimensional space. The diversity information can be decomposed into within- and between- samples diversity values. This allowed the use of a "pseudo F" (1) statistic (the ratio of within-cluster diversity and between-cluster diversity) to examine possible clustering phenomena, and significance was evaluated by permutation tests. The P test (2, 3) also was used to assess for significant differences between samples.1. Hughes JB, Hellmann JJ, Ricketts TH, Bohannan BJ (2001) Appl Environ Microbiol 67:4399-4406.
2. Lozupone C, Hamady M, Knight R (2006) BMC Bioinformatics 7:371.
3. Martin AP (2002) Appl Environ Microbiol 68:3673-3682.