Metagenomic sequencing of unknown tropical febrile illness cases
Total nucleic acid from 140 microl of serum was extracted using the QIAamp Viral RNA Isolation Kit (Qiagen), which co-purifies RNA and DNA. End-tagged dsDNA libraries were created as follows. RNA was reverse transcribed in reactions containing 1× reaction buffer, 5 mM dithiothreitol, 1.25 mM dNTPs, 20 pmoles primer (5'-CGC TCT TCC GAT CTN NNN NN-3'), 100 U Superscript III (Invitrogen), and ~20 ng template. Following reverse transcription, Sequenase reaction buffer and 2 U of Sequenase DNA polymerase (Affymetrix) were added to samples for second strand synthesis. The Sequenase reactions were performed twice so that starting DNA templates would be converted into end-tagged library molecules. The resulting libraries were amplified by PCR using primer 5'-CGC TCT TCC GAT CT-3'. PCRs contained 1× reaction buffer, 2 µM primer, 0.25 mM dNTPs, 2 U Taq DNA polymerase, and 2 µl library template. Thermocycling conditions were 95°C for 2 min; 25 cycles of 95°C for 30 sec, 40°C for 30 sec, and 72°C for 1 minute, with a final extension of 5 minutes.   The Illumina paired-end adapter sequences were appended to library molecules using PCR. Library generation primers are listed in Table S1 of the accompanying publication (PubMed ID 22347512) were modified from adapter A and adapter B sequences (Illumina).  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 3'-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 94°C for 30 s, 40°C for 30 s, and 72°C for 1 min, followed by 15 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C 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 3'-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.