Figure 1.
Deep sequencing strategy to determine abundance and mutation linkage of NS3 RAVs.
(A) Roche/454 pyrosequencing. Primer binding sites for 454/Roche pyrosequencing primers are shown in yellow. The pyrosequencing primers are composites containing the required sequences for the Roche/454 titanium chemistry procedure at 5′ end (blue and green), a unique 8-base DNA barcode that indexes each sample (red), and HCV-specific primer sequences at the 3′ end (yellow). (B) Illumina paired-end sequencing. Partial NS3 gene segments were first amplified using gene-specific primers that contain HCV-specific sequences (yellow), a barcode sequence unique to each sample (red) and partial adapter sequences (green and blue). Amplified fragments were tailed with flow cell adaptors (green and blue arrows), then subjected to paired-end sequencing using the standard Illumina paired-end protocol. This protocol will generate non-overlapping forward (100 nt) and the reverse (100 nt) reads from each cluster, providing precise long-range positional and sequence information. After trimming and quality control, the paired-end reads allowed linkage analysis between V36-V55 and R155-I170. Sequences of all primers used in (A) and (B) are listed in Supporting Information S1.
Table 1.
Demographics, viral load, and time points of the samples used in this study.
Figure 2.
Frequency of NS3 resistance-associated variants (RAVs) in (A) chronic HCV and (B) longitudinal liver transplant recipients as determined by Roche/454 pyrosequencing.
The proportion of pyrosequencing reads that harbored authentic RAVs is indicated by the intensity of magenta shown in the heatmap (p≤0.05, chi-square test). RAVs detected in pyrosequence reads that were not significantly enriched compared to background technical error rates are shown in grey (p>0.05, chi-square test). The background error rate was determined using in vitro control transcripts of known sequence that were amplified and sequenced in parallel with the RNA extracted from HCV subjects. Each column is a different sample, and each row represents a different RAV detected by pyrosequencing. Asterisks denote RAVs detected by direct Sanger sequencing. In (A), each column is a different subject (14 genotype 1a and 6 genotype 1b), and in (B), longitudinal samples within each subject (as indicated by the subject code at the bottom) are ordered from left to right, and phylogenetic analysis demonstrates that temporally associated samples are more closely related within subjects than samples between subjects.
Figure 3.
Dynamics of major NS3 variants in liver transplant recipients.
Forward pyrosequence reads (∼337 nt, corresponding to coordinates 3342 to 3674 on the H77 genome, accession NC_004102) were clustered into operational taxonomic units (OTUs) at 97% sequence identity. The relative abundance of each major variant is shown by the black shading, where the extent of the black region from left to right within the gray bar indicates the proportion in the total viral population. The black double-headed arrow denotes the time of liver transplantation, and the number of days before and after liver transplantation are indicated at the bottom. Trees were generated using UPGMA. Shannon diversity values based on NS3 pyrosequence reads represent changes in NS3 diversity over time (Top panels). ALT: alanine aminotransferase. (A) The major variant that established re-infection post-LT was identical or closely related to the dominant variant pre-LT (B) Two minor variants pre-LT became dominant quasispecies post-LT.
Table 2.
Mutation linkage analyzed by Illumina paired-end sequencing.
Table 3.
Illumina paired-end sequencing identifies low frequency T54A+R155K double mutant in mock RNA populations.