Computational protocol: A novel standardized deep sequencing-based assay for hepatitis C virus genotype determination

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Protocol publication

[…] Briefly, total RNA was extracted from 400 µL of serum or plasma by means of QIAsymphony DSP Virus/Pathogen kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer’s instructions. The RNA pellet was eluted with 60 µL of RNAse-free water with 0.04% NaN3 and stored at −20 °C until analysis. One-step reverse transcriptase (RT)-PCR was performed with 15 µL of total extracted RNA using the QIAGEN OneStep RT-PCR kit, according to the manufacturer’s instructions. A nested PCR technique was used to amplify an NS5B-coding DNA fragment. The first round used external sense and antisense primers Sn755 and Asn1121 and consisted of 40 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min. The second round used internal sense primer NS5B-SI766 and antisense primer NS5B-ASI1110 and consisted of 35 cycles at 95 °C for 30 s, 58 °C for 30 s, and 68 °C for 1 min. PCR products were purified by means of NucleoFast 96 PCR plate kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) and directly sequenced by means of the BigDye Terminator Cycle v3.1 sequencing kit (ThermoFisher Scientific, Courtabœuf, France) on an ABI 3100 sequencer (Applied Biosystems, Foster City, California), according to the manufacturer’s instructions. Phylogenetic analysis was carried out using genotypes 1 to 7 reference sequences available in GenBank, by means of the Phylogeny Inference Package (PHYLIP), version 3.695. Nucleotide sequences (nucleotide position 724–1009 according to the H77-1a prototype strain) were aligned with the reference sequences using CLUSTAL W. Phylogenetic relationships were deduced by means of DNADIST-NEIGHBOR from PHYLIP. For neighbor-joining analysis, a Kimura 2-parameter distance matrix with a transition/transversion ratio (Ts/Tv) of 2.0 was used. Phylogenetic trees were plotted with FigTree v1.4.3. Their robustness was assessed by bootstrap analysis of 1,000 replicates by means of the SEQBOOT program from PHYLIP. For recombinant 2k/1b strains, the HCV E1 region was amplified and sequenced in addition to the NS5B region, as already described. [...] Briefly, nucleic acid extraction was performed from 530 µL of serum on the Sentosa SX101 robotic instrument using Sentosa Virus Total Nucleic Acid Plus II kit. The NS3, NS5A and NS5B coding regions were RT-PCR amplified by means of Veriti Dx 96-Well Thermal Cycler (Applied Biosystems). After purification of PCR products via magnetic beads, a 200-nucleotide fragment library was prepared on Sentosa SX101. The samples were barcoded by ligation, pooled into a single tube and amplified by emulsion PCR on Sentosa ST401i. Deep sequencing was performed by means of the Sentosa SQ Sequencing Kit on the Sentosa SQ301 Sequencer, based on Ion Torrent technology. Primary data analysis was automatically performed using Sentosa SQ Reporter software. Assembled NS5B contigs (a 685-base pair fragment) were aligned to all NS5B reference sequences using Basic Local Alignment Search Tool (BLAST) and phylogenetic analysis was automatically performed by the software. Based on the manufacturer’s claim, the minimum amount of HCV RNA needed for HCV genotype/subtype determination in the assay is 1,000 IU/mL for genotypes 1a, 1b, 2, 3 and 4 and 2,000 IU/mL for genotypes 5 and 6. For subtypes 1a and 1b differentiation, a 944-base pair fragment in the NS3 region and a 604-base pair fragment in the NS5A region were also sequenced and analyzed by the system. Subtype 1a and 1b sequences were aligned and analyzed through a similar process. […]

Pipeline specifications

Software tools PHYLIP, Clustal W, FigTree, BLASTN
Application Phylogenetics
Organisms Homo sapiens