Genomewide pyrosequencing of HCV-1a, with analysis of intrahost variability. (A) Schematic representation of HCV-1a amplification strategy and sequence coverage obtained for each patient. Gray bars denote RT-PCR amplicons relative to the HCV-1a genome. The nucleotide numbering is based on the sequence of HCV strain H77 (GenBank accession no. NC_004102). (B) Graphs indicating the SNP frequency for each patient along the consensus genomic sequence. Synonymous mutations are indicated in black, and nonsynonymous mutations are shown in red. The HCV-1a genomic organization is depicted at the top of the figure. The nucleotide numbering is based on the sequence of HCV strain H77 (GenBank accession no. NC_004102).

Genomewide pyrosequencing of HCV-2b/1a DNA clone in order to determine error rates associated with PCR and Roche/454 pyrosequencing. (A) Sequence coverage obtained across the HCV-2b/1a genome. The HCV-2b/1a genomic organization is depicted at the top of the figure. The nucleotide numbering is based on the sequence of HCV strain H77 (GenBank accession no. NC_004102). (B) Graph indicating the SNP frequency across the HCV-2b/1a genome on a 50% y axis (top) and 1% y axis (bottom). The HCV-2b/1a genomic organization is depicted at the top of the figure. The nucleotide numbering is based on the sequence of HCV strain H77 (GenBank accession no. NC_004102).

Phylogenetic tree showing evolutionary relationships between 382 complete HCV-1a genome sequences. A maximum likelihood phylogenetic tree is presented that demonstrates the separation of the HCV-1a isolates into two distinct clades: clade 1 (black) and clade 2 (blue). The four consensus sequences generated in this study (for patients A to D) are highlighted in red and grouped in clade 1.