For deep sequencing, the Illumina paired-end adapter sequences were appended to library molecules using PCR, essentially as previously described [28] . Library generation primers (Table S1) were modified from adapter A and adapter B sequences (Illumina). Samples were reverse transcribed and libraries were created and amplified as described above for the Virochip. Library molecules of approximately 300 bp were purified on a 4% native polyacrylamide gel, ethanol precipitated, and PCR amplified for 17 additional cycles using a 22-nt-long primer consisting of the 39-end of Illumina adapter A (primer 2) and the full-length 61-bp Illumina adapter B (primer 4) under the following conditions: 2 cycles of 94uC for 30 s, 40uC for 30 s, and 72uC for 1 min, followed by 15 cycles of 94uC for 30 s, 55uC for 30 s, and 72uC for 1 min. Amplicons generated with the correct adapter topology (one end with adapter A and the other with adapter B) were approximately 355 bp and were separated by polyacrylamide gel electrophoresis from adapter A/A and adapter B/B amplicons, which migrate differently (approximately 40 bp smaller or larger than the expected size). An additional 10 cycles of PCR were then performed using the full-length adapter sequences as primers (primers 3 and 4). Libraries were validated by Sanger sequencing before high throughput sequencing. Following validation, samples were combined into five pools for sequencing. For pools one through four reverse transcription primers included a three or four-nucleotide barcode sequence at the 39-end. For pool five, barcodes were located internally in the adapter sequence. Each pool was sequenced on one lane of a flowcell on the Illumina Genome Analyzer II (pools 1-4) or HiSeq 2000 (pool 5). Pools' 1-4 molecules were sequenced as 67 nucleotide paired ends, and pool 5 molecules as 97 nucleotide paired ends. Paired-end sequencing was performed for several reasons: (1) to double the overall amount of data generated, (2) to double the amount of sequence information per molecule, and (3) to provide anchors from which additional sequence could be recovered by subsequent PCR.