The genotyping of each DNA sample was performed by real-time PCR with a model 7500 sequencer (ABI, Tokyo, Japan) using FAM- and VIC-labeled single nucleotide (nt) polymorphism (SNP) probes for the locus rs8099917 (ABI). Deep sequencing of part of the viral NS5A region was performed for each of the 110 patients. Briefly, RNA was extracted from the stored sera and reverse transcribed to complementary DNA.[23] Then, two-step nested PCR was carried out with primers specific for the NS5A region of the HCV genome. To avoid PCR selection bias, we searched for the most conserved DNA sequence

regions around NS5A by examining sequence information published previously from 43 HCV positive individuals from Japan[16] and designed novel primers for this study 5-Fluoracil cost (Supplementary Table 1). This PCR procedure amplified 436 viral nt, including the 1st to 432nd nt of the NS5A region. The primers for the second-round PCR had barcodes, 10 nt in length, attached and these differed for each sample, so that the PCR products

from each sample were identifiable. After the band densities of the PCR products were quantified using a Pico Green dsDNA Assay Kit (Invitrogen), the concentrations of the samples were adjusted to a common value and pooled samples were prepared. Libraries were then subjected to emulsion PCR, the enriched DNA beads were loaded onto a picotiter plate and pyrosequencing was carried out with a Roche GS Junior/454 U0126 ic50 sequencing system using titanium chemistry (Roche, Branford, CT, USA). The Roche Variant Analyzer version 2.5pl (Roche) was used for the analysis. Statistical differences in the parameters, including all available patients’ demographic, biochemical, hematological, Rutecarpine virological and SNP data in the three groups (naïve, relapser and null responder), classified according to the response to previous PEG IFN/RBV therapy, were determined using the χ2-test for categorical variables and Kruskal–Wallis test for numerical variables. Statistical differences in the parameters in two groups (Y93H positive, Y93H negative) were determined by Student’s

t-test or Mann–Whitney U-test for numerical variables and Fisher’s exact test or χ2-test for categorical variables. Variables that achieved statistical significance (P < 0.05) in univariate analysis were entered into multiple logistic regression analysis to identify significant independent factors. We also calculated the odds ratios and 95% confidence intervals. All P-values of less than 0.05 by the two-tailed test were considered significant. To perform deep sequencing analysis of the NS5A region from many patients, simultaneous analysis was carried out using the barcode primers and approximately 3826 reads were obtained per sample from each group of patients (naïve, relapser and null responder) (Table 2).