Faststart high fidelity taq dna polymerase

HVR1 amplification was performed from pretreatment serum samples as described previously
[26 (link)]. In brief, viral RNA was extracted from 250 μl of serum by modified guanidinium thiocyanate-phenol/chlorophorm method, then subjected to reverse transcription at 37°C for 30 minutes using AccuScript High Fidelity Reverse Transcriptase (Agilent Technologies). A fragment of E2 region containing HVR1 was amplified in two-step PCR using FastStart High Fidelity Taq DNA Polymerase (Roche). Primers for the second round PCR contained tags recognized by GS Junior sequencing platform, standard 10-nucleotide multiplex identifiers (MID) and target-specific sequence.

We used the SMARTer-RACE cDNA amplification kit (Clontech) to create RACE cDNA by reverse transcription of 1 μg total RNA extracted from the dorsal gland tissue. To amplify the full coding sequence of the most abundant SPF isoforms, primers were designed on the 3′ untranslated region of the highly expressed SPF isoforms obtained from the RNA sequencing procedure (see above). In addition, eleven degenerate primers from a previous study21 were used to obtain a large diversity of SPF protein sequences (Table S1). PCR products were amplified with FastStart High Fidelity Taq DNA polymerase (Roche) using a wide range of annealing temperatures. The following PCR conditions were used: one initial denaturation for 240 s at 94 °C, followed by 36 cycles with denaturation for 40 s at 94 °C, annealing for 60 s at different temperatures, and elongation for 60 s at 72 °C. To clone these amplification products, we used a pGEM-T Easy cloning vector (Promega). Vectors were transformed into TOP10 Competent Cells (Invitrogen). Colonies were picked randomly and inserts were amplified with Faststart Taq DNA polymerase using the same above-mentioned conditions. Amplification products (96 in total) were purified and sequenced, and sequence editing was performed with CodonCode Aligner version 3.7.1.1. Contigs were constructed with 99% identical bases and translated into amino acid sequences.

HVR1 amplification was performed as described previously^{27 (link)}. In brief, total RNA was extracted from 250 μl of plasma by a modified guanidinium thiocyanate-phenol/chlorophorm method using Trizol (Life Technologies, Carlsbad, CA, USA). RNA was subjected to reverse transcription at 37 °C for 30 minutes using AccuScript High Fidelity Reverse Transcriptase (Agilent Technologies, Santa Clara, CA, USA) and random hexamers (Life Technologies). A gene fragment encompassing HVR1 was amplified in two-step PCR using FastStart High Fidelity Taq DNA Polymerase (Roche, Indianapolis, IN, USA) using target-specific primers (Table 2). Additionally, primers employed in the second round PCR contained tags recognized by GS Junior sequencing platform and standard 10-nucleotide multiplex identifiers^{27 (link)}.Table 2

Primer sequences employed in the study.

	genotype 1b HVR1 amplification	Positions of HCV genome	genotype 3 HVR1 amplification	Positions of HCV genome
First round PCR	Forward: 5′-GGTGCTCACTGGGGAGTCCT-3′	1389–1408	Forward: 5′-ATGGCATGGGATATGAT-3′	1291–1307
	Reverse: 5′-CATTGCAGTTCAGGGCCGTGCTA-3′	1632–1610	Reverse: 5′-AAGGCCGTCCTGTTGA-3′	1619–1604
Second round PCR	Forward: 5′- TCCATGGTGGGGAACTGGGC-3′	1428–1447	Forward: 5′-GGCAACTGGGCCAAGGTCGC-3′	1437–1456
	Reverse: 5′-TGCCAACTGCCATTGGTGTT-3′	1603–1584	Reverse: 5′-ATGTGCCACGAGCCATTGGT-3′	1606–1587