Human metagenome isolated from blood sample of patient CHC_006 with chronic HCV infection
Design description  DNA was extracted from blood sample, and PCR amplification of the V2 region of bacterial 16S rRNA genes and pyrosequencing were preformed.  Library Construction protocol:  Triplicate PCR reactions were performed for each sample. The forward primer (5’- CTATGCGCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG- 3’) contained the 454 Life Sciences primer B sequence, the broadly conserved bacterial primer 27F, and a two-base linker sequence (“TC”). The reverse primer (5’- CGTATCGCCTCCCTCGCGCCATCAGNNNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’) contained the 454 Life Sciences primer A sequence, a unique 10-nt barcode used to tag each PCR product (designated by NNNNNNNNNN and provided by Roche), the broad-range bacterial primer 338R, and a “CA” linker sequence inserted between the barcode and the rRNA primer. Each 25 μl reaction contained 0.2 μM forward and reverse primers, 3 μl template DNA, and 2.5 ul 10X PCR buffer plus Mg2+ (TaKaRa), 2.0 ul dNTP (2.5 mM each) (TaKaRa), 0.75 ul DMSO (100%), 0.25 ul TaKaRa TaqTM (5 U/μl). All dilutions were carried out using sterile ddH2O. PCR reactions were assembled within a PCR hood in which all surfaces and pipettes had been decontaminated with 30 min autoclaving at 120℃ and UV irradiated for 30 minutes. Thermal cycling was 95°C for 10 minutes, followed by 30 cycles of 95°C for 30 seconds, 52°C for 30 seconds, and 72°C for 90 seconds, with a final extension of 10 minutes at 72°C. Replicate amplicons were pooled and visualized on 1.5% agarose gels using EB stain in 0.5X TE. Amplicons were cleaned using MinElute® Gel Extraction Kit (QIAGEN) according to the manufacturer’s instructions. Amplicon DNA concentrations were determined using the Quant-iT PicoGreen dsDNA reagent and kit (Invitrogen). Using the amplicon pool, we carried out pyrosequencing using primer A and Titanium chemistry on a 454 Life Sciences Genome Sequencer FLX instrument (Roche) at the DNA Sequencing Facility at the Kunming Institute of Zoology, Chinese Academy of Sciences.