Total nucleic acid from 140 ml of serum was extracted using the QIAamp Viral RNA Isolation Kit (Qiagen), which co-purifies RNA and DNA. End-tagged dsDNA libraries were created essentially as previously described [28] . RNA was reverse transcribed in reactions containing 16 reaction buffer, 5 mM dithiothreitol, 1.25 mM dNTPs, 20 pmoles primer (59-CGC TCT TCC GAT CTN NNN NN-39), 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 59-CGC TCT TCC GAT CT-39. PCRs contained 16 reaction buffer, 2 mM primer, 0.25 mM dNTPs, 2 U Taq DNA polymerase, and 2 ml library template. Thermocycling conditions were 95uC for 2 min; 25 cycles of 95uC for 30 sec, 40uC for 30 sec, and 72uC for 1 minute, with a final extension of 5 minutes. These libraries were further processed for microarray hybridization and deep sequencing as described below.