Confirmatory sequencing of full-genome amplicons generated by PCR.

<p>A. For each amplicons, the sequencing was carried out with primers other than those used for full-genome amplification. The linear sequencing template (black line), the PCR primers are shown at both ends of the amplicons. Bleu lines (amplicons 671F-892R), green lines (amplicons 6838F-6972R), red lines (amplicons 6838F-7076R) represent the sequenced regions attached to the corresponding sequencing primers. Numbers included in primer IDs represent the position relative to HPV_SD2 genome. B. Alignment of sequences generated by Sanger method along the HPV_SD2 generated by 454 sequencing. Bars in blue, green and red correspond to sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058404#pone-0058404-g006" target="_blank">Figure 6A</a>. The newly generated sequences covered 4,622 nucleotides representing 63.3% of the total HPV_SD2 genome. C. Comparison of the sequences generated by Sanger method and HPV_SD2 contig generated by 454 sequencing. Size of each Sanger sequence and percentage identity are shown.</p

Summary of known viruses detected in nasopharyngeal samples.

<p>Summary of known viruses detected in nasopharyngeal samples.</p

Taxonomical assignment of sequence reads convenient respiratory waste samples.

<p>A. Overall proportions of sequences with homolog in Genbank (the known, 7.8%) compared to sequences with no homolog in Genbank (Unknown, 92.2%). Unknown/Divergent indicates the proportion of highly divergent and/or novel sequences with no homology to NCBI. B. Proportion of sequences classified as eukaryotes, bacteria and viruses. Sequences were classified using BLASTn search against all non-redundant nucleotide sequences in the NCBI nt database with an E-value cutoff of 10⁻⁵.</p

Composition of sample pools and methods for DNA and RNA extraction.

<p>Composition of sample pools and methods for DNA and RNA extraction.</p

Coverage plot of the circular structure of HPV-SD2.

<p>A. Screenshot of the assembly of sequences using the <i>De Novo Assembler</i> set for 98% similarity and 45 bp overlap. The coverage plot is shown in green. Overhanging sequences at the 5′ and 3′ ends are partially assembled. B. Box at 5′ end and box at 3′ end are highlighted to show the similarity between unassembled overhang reads at 5′ and 3′ ends and corresponding ends of the consensus sequence.</p

Genomic organization of the HPV_SD2 virus.

<p>Open reading frames (L2, L1, E6, E7, E1, E2, E4) and a 530 bp non-coding long control region (LCR) are shown. B. Details of the LCR region showing the TATA box (TATAAA, positions 3735–3740), a polyadenylated site (AATAAA, positions 3336–3341), 3 palindrome sites (ACCG-N₄-CGGT; positions 3483–3494, 3650–3661, 3720–3731) and 1 degenerate palindrome (ACC-N₆-GGT, positions 3524–3535). C. Metal-binding domains in deduced E6 and E7 proteins.</p

Maximum likelihood tree showing the clustering of HPV_SD2 with previously documented full-length genomes of human papillomaviruses.

<p>The tree was visualized with the Interactive Tree of Life (iTOL) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058404#pone.0058404-Letunic1" target="_blank">[32]</a>. Bootstrap values with at least 50%, 75% or 100% (100 re-samplings) are indicated. Note, another HPV_SD1 from this study was added in this analysis clustered with reference HPV_49.</p

Confirmatory PCRs to verify HPV_SD2 is a circular double-stranded genome.

<p>A. Graphical representation of the binding sites of primers 671F-892R on the putative circular structure of HPV_SD2, and the predicted PCR product sizes (I: 222 bp, II: 7,591 bp, III: 14,890 bp). The predicted short band (I) indicates the amplification of the proximal region between primers 671F and 892R. The large band (II) indicates that <i>Taq</i> DNA polymerase would amplify the region between the primers 671F and 892R by making the full circle of the HPV genome. The large band (III) indicates the <i>Taq</i> DNA polymerase would make 2 full circle around the HPV genome. PCRs were also performed using primer sets 6838F-6972R, 6838F-7076R. B. Agarose gel (0.5%) showing the amplified HPV_SD2. Primer sets used are shown: primer sets 6838F-6972R, 6838F-7076R. For each PCR, the same sample pool was tested at different concentrations [1∶1 (lanes 1, 4, 7), 1∶10 (lanes 2, 5, 8) and 1∶50 (lanes (3, 6, 9)] using 1 µl per reaction. L: DNA ladder. Amplicons can be seen at the expected sizes.</p

Location of the 29 islands surveyed.

<p>Color scale indicates oceanic net primary production derived from satellite data using the Vertically Generalized Production Model (VGPM). Circles indicate the relative NCEAS cumulative human impact score for each island. For island abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043233#pone-0043233-t001" target="_blank">Table 1</a>.</p

Survey data and calculated values for 29 islands in the Pacific, grouped by region.

<p>Predicted metabolic rates are basal rates. NPP = net primary production. Colors identify each island group in the figures.</p