The same PCR products obtained previously for the Sanger sequencing of the NS3/4 A, NS5A and NS5B regions were analyzed using a next generation sequencing (NGS) approach. Samples from 15 DAA failing patients as well as 15 untreated patients with at least 10000 HCV copies/ml were analyzed by NGS.
HCV amplicons (20 µL) were purified using 36 µL Agencourt AMPure XP beads (Beckman Coulter Inc). Amplicon concentrations were determined with the Invitrogen™ Quant-iT™ PicoGreen® dsDNA Assay (Invitrogen, Inc.). Purified amplicons were diluted to 5.0 ng/µl and 2 µL were processed using the Illumina Nextera XT DNA Library Preparation Kit (Illumina Inc.) and uniquely indexed using the Illumina Nextera XT Index Kit (96 Index). All amplified Nextera-PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter Inc.). All recovered DNA was pooled in equal volumes at a normalized concentration (1.43 ng/µl, the lowest concentration recovered). The DNA concentration of the pool was determined using the Invitrogen™ Quant-iT™ PicoGreen® dsDNA Assay (Invitrogen,Inc.). Library quality was determined using a Bioanalyzer 2100 (Agilent Technologies) and was sequenced on a MiSeq deep sequencing platform (Illumina, San Diego, CA) using a the 500-cycle MiSeq reagent kit v2.Fastq files were generated using the onboard MiSeq Reporter.
HCV amplicons (20 µL) were purified using 36 µL Agencourt AMPure XP beads (Beckman Coulter Inc). Amplicon concentrations were determined with the Invitrogen™ Quant-iT™ PicoGreen® dsDNA Assay (Invitrogen, Inc.). Purified amplicons were diluted to 5.0 ng/µl and 2 µL were processed using the Illumina Nextera XT DNA Library Preparation Kit (Illumina Inc.) and uniquely indexed using the Illumina Nextera XT Index Kit (96 Index). All amplified Nextera-PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter Inc.). All recovered DNA was pooled in equal volumes at a normalized concentration (1.43 ng/µl, the lowest concentration recovered). The DNA concentration of the pool was determined using the Invitrogen™ Quant-iT™ PicoGreen® dsDNA Assay (Invitrogen,Inc.). Library quality was determined using a Bioanalyzer 2100 (Agilent Technologies) and was sequenced on a MiSeq deep sequencing platform (Illumina, San Diego, CA) using a the 500-cycle MiSeq reagent kit v2.Fastq files were generated using the onboard MiSeq Reporter.