Computational protocol: High resolution sequencing of hepatitis C virus reveals limitedintra-hepatic compartmentalization in end-stage liver disease

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Protocol publication

[…] RT-PCR amplification was performed across the HCV genome using an overlapping amplicon approach to generate near full-length genomes for deep sequencing. For genotype 1a, the first amplicon encompassed the structural proteins from core to NS2 (H77: 279–3542), the second spanned from E2 to NS4B (2290–4774) and the third from NS3 to NS5A (4656–7148). A fourth amplicon covering the remainder of NS5A and the NS5B region failed to amplify. For genotype 3a, the first, second and fourth amplicons were successfully amplified (279–3542, 2486–5776 and 7373–9365 (NS5A to NS5B)) whereas amplicon 3, covering the remainder of NS4B and the 5′half of NS5B, failed amplification. For each amplicon 1–200 ng of hepatic RNA (∼1000 HCV copies) or RNA extracted from 100 μl of plasma (∼10,000 HCV copies) was used as input template. For amplicon 1, the reaction consisted of sense (177s: CCT TGT GGT ACT GCC TGA TAG) and antisense primers (3542a-1a: GGG YAG CAG TTG ACA CRA TCT or 3542a-3a: CTG GGT AGC CGT AGA AAG CAC CT) at 0.4 μM, and a Superscript III RT/Platinum Taq Mix in the manufacturers supplied First Strand Buffer (Invitrogen), with the following conditions: cDNA synthesis for 30 min at 55 °C followed by heat denaturation at 94 °C for 2 min, and PCR amplification conditions of 40× (94 °C, 15 s; 58 °C, 30 s; 68 °C 240 s), with a final extension at 68 °C for 10 min. For amplicons 2 and 3 from genotype 1a, this reaction consisted of sense (A2F: AAC GTT GCG ATC TGG AAG AC or A3F: GCT CTC ATG ACC GGC TTT AC) and antisense primers (A2R: GGA AGC GTG GTT GTC TCA AT or A3R: AGA GAT CTC CCG CTC ATC CT) with the same reaction reagents as before but with an adjustment to the PCR amplification conditions, which were 40× (94 °C, 15 s; 55 °C, 30 s; 68 °C 180 s), with a final extension at 68 °C for 5 min. For amplicons 2 and 4 from genotype 3a the primers were : sense (08F: TGG GAT GGG CGY TGA ART GG or 21F: ATG TGT CYG CRG CGC TAG C) and antisense (15R: TAG TTT GGT TGG TCG TCA GG or 25R: AGT AGG AGT AGG CAA AGC AGC) at 0.4 μM, with the same reaction reagents as before but with the following reaction conditions; for amplicon 2: cDNA synthesis for 30 min at 55 °C, heat denaturation at 94 °C for 2 min, then PCR of 40× (94 °C, 15 s; 55 °C, 30 s; 68 °C 180 s), with a final extension at 68 °C for 10 min. For amplicon 4 the PCR conditions were 40× (94 °C, 15 s; 64 °C, 30 s; 68 °C 180 s), with a final extension at 68 °C for 10 min. All PCR products were visualized on 1% agarose gels and purified using the PureLink Quick Gel Extraction Kit (Invitrogen).For deep sequencing, the PCR amplicons were fragmented and barcoded using NexteraXT DNA Library Prep Kit, as per the manufacturer’s protocol. Samples were pooled and sequenced on an Illumina MiSeq platform, using a 2 × 250 bp V2 reagent kit. Paired-end reads were assembled into a HCV consensus sequence using the VICUNA de novo assembler software and finished with V-FAT v1.0. Reads were mapped back to the consensus using Mosaik v2.1.73, and intra-host variants called by V-Phaser v2.0 , . All reads have been deposited to the NCBI Sequence Read Archive under the study number SRP065844. […]

Pipeline specifications

Software tools VICUNA, V-FAT, MOSAIK
Databases SRA
Application qPCR
Organisms Homo sapiens, Human poliovirus 1 Mahoney
Diseases Hepatitis C, Hepatitis C, Chronic, End Stage Liver Disease