HVR1 Amplification from Serum RNA

HVR1 amplification was done as described in a previous publication [20 (link)]. In brief, total RNA was extracted from 250 μl of serum by a modified guanidinium thiocyanate-phenol/chlorophorm method using Trizol (Life Technologies, Carlsbad, CA, USA). Next, RNA was subjected to reverse transcription at 42°C for 60 minutes using AccuScript High Fidelity Reverse Transcriptase (Agilent Technologies, Santa Clara, CA, USA) and random hexamers. A region of 175 nt length encompassing HVR1 was amplified in two-step PCR using FastStart High Fidelity Taq DNA Polymerase (Roche, Indianapolis, IN, USA). Primers used for the first round amplification were as follows: 5′-CATTGCAGTTCAGGGCCGTGCTA-3′ (nt 1632–1610) and 5′-GGTGCTCACTGGGGAGTCCT-3′ (nt 1389–1408), according to the sequence of reference strain H77 (GenBank accession no. AF009606). Primers employed in the second PCR contained tags recognized by GS Junior sequencing platform, standard 10-nucleotide multiplex identifiers and target-complementary sequence [5′- TCCATGGTGGGGAACTGGGC-3′ (positions 1428–1447) and 5′-TGCCAACTGCCATTGGTGTT-3′ (positions 1603–1584)] [20 (link)].