Computational protocol: An NS5A single optimized method to determine genotype, subtype and resistance profiles of Hepatitis C strains

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Protocol publication

[…] In order to amplify the NS4B-NS5A region, different degenerate primers were designed (). 293 HCV NS4B-NS5A sequences (147 G1 and 146 G2 to G5) extracted from databases (GenBank and the Los Alamos National Laboratory) were aligned with SeqScape software (version 2.5, Applied Biosystems) which enabled the identification of a segment (6066–6882 nucleotides) for the design of consensus primers. We used a unique sense primer NS5A-2F (26 nucleotides including 6 degenerate positions). In order to improve the PCR regardless of the genotypes 1 to 5, two reverse primers were chosen and used together: NS5A-R for G1,G2, G4, G5 and NS5A-R3 for G3, located at the same region. They are used together (25 nucleotides including 8 degenerate positions). To facilitate the sequencing reaction, the M13 universal primers were added to the 5 'end of each PCR primer. [...] To investigate the suitability of the nucleotide NS4B-NS5A sequence fragment for genotyping, NS4B-NS5A fragments from complete reference genomes provided in Smith et al [] were aligned and a phylogenetic analysis was performed using the Neighbor-Joining method (MEGA5 software, []) The reliability of the phylogenetic clustering was evaluated using bootstrap analysis with 1000 replicates (). The 142 sequences from the clinical samples were aligned with the same confirmed sequences and a phylogenetic tree was built using identical parameters (). The type and subtype were compared to those obtained by sequencing the NS3 fragment. […]

Pipeline specifications

Software tools SeqScape, MEGA
Applications Phylogenetics, Sanger sequencing
Organisms Classical swine fever virus, Homo sapiens