Fig 3.

Detection and single-stranded nature of RNA viral sequences within hot springs months after sampling for metagenomic analysis. Total nucleic acids of samples obtained 18 months after the original sampling were extracted from either the viral fraction or total cellular fraction of NL10 (B) and NL18 (A), DNase treated, and subjected to RT-PCR for metagenomic analysis. The detection and strand-specific nature of contig00009 (A) and contig00002 (B) were determined using contig-specific primer sets (see Table S2 in the supplemental material). Either forward (+), reverse (−), or both (+/−) primers were added to the initial first-strand cDNA synthesis mix and the mixture incubated as described in Materials and Methods. After first-strand synthesis, the complete primer pair was added (if necessary) for the PCR stage. The control, where reverse transcriptase was excluded from the procedure (−RT), and the molecular-size marker (bp) are indicated.