Deep Sequencing Analysis of HCV NS3 Resistance-Associated Variants and Mutation Linkage in Liver Transplant Recipients

Viral variants with decreased susceptibility to HCV protease inhibitors (PIs) occur naturally and preexist at low levels within HCV populations. In patients failing PI monotherapy, single and double mutants conferring intermediate to high-level resistance to PIs have been selected in vivo. The abundance, temporal dynamics and linkage of naturally occurring resistance-associated variants (RAVs), however, have not been characterized in detail. Here, using high-density pyrosequencing, we analyzed HCV NS3 gene segments from 20 subjects with chronic HCV infection, including 12 subjects before and after liver transplantation. Bioinformatics analysis revealed that Q80 substitution was a dominant variant in 40% of the subjects, whereas other RAVs circulate at low levels within quasispecies populations. Low frequency mutation linkage was detectable by Illumina paired-end sequencing in as low as 0.5% of the mock populations constructed from in vitro RNA transcripts but were uncommon in vivo. We show that naturally occurring RAVs are common and can persist long term following liver transplant at low levels not readily detectable by conventional sequencing. Our results indicate that mutation linkage at low levels could be identified using the Illumina paired-end approach. The methods described here should facilitate the analysis of low frequency HCV drug resistance, mutation linkage and evolution, which may inform future therapeutic strategies in patients undergoing direct acting antiviral therapies

Coordinated Genetic Regulation of Growth and Lignin Revealed by Quantitative Trait Locus Analysis of cDNA Microarray Data in an Interspecific Backcross of Eucalyptus

Phenotypic, genotypic, and transcript level (microarray) data from an interspecific backcross population of Eucalyptus grandis and Eucalyptus globulus were integrated to dissect the genetic and metabolic network underlying growth variation. Transcript abundance, measured for 2,608 genes in the differentiating xylem of a 91 (E. grandis × E. globulus) × E. grandis backcross progeny was correlated with diameter variation, revealing coordinated down-regulation of genes encoding enzymes of the lignin biosynthesis and associated methylation pathways in fast growing individuals. Lignin analysis of wood samples confirmed the content and quality predicted by the transcript levels measured on the microarrays. Quantitative trait locus (QTL) analysis of transcript levels of lignin-related genes showed that their mRNA abundance is regulated by two genetic loci, demonstrating coordinated genetic control over lignin biosynthesis. These two loci colocalize with QTLs for growth, suggesting that the same genomic regions are regulating growth, and lignin content and composition in the progeny. Genetic mapping of the lignin genes revealed that most of the key biosynthetic genes do not colocalize with growth and transcript level QTLs, with the exception of the locus encoding the enzyme S-adenosylmethionine synthase. This study illustrates the power of integrating quantitative analysis of gene expression data and genetic map information to discover genetic and metabolic networks regulating complex biological traits

Upper versus lower airway microbiome and metagenome in children with cystic fibrosis and their correlation with lung inflammation.

ObjectiveAirways of children with cystic fibrosis (CF) harbor complex polymicrobial communities which correlates with pulmonary disease progression and use of antibiotics. Throat swabs are widely used in young CF children as a surrogate to detect potentially pathogenic microorganisms in lower airways. However, the relationship between upper and lower airway microbial communities remains poorly understood. This study aims to determine (1) to what extent oropharyngeal microbiome resembles the lung microbiome in CF children and (2) if lung microbiome composition correlates with airway inflammation.MethodThroat swabs and bronchoalveolar lavage (BAL) were obtained concurrently from 21 CF children and 26 disease controls. Oropharyngeal and lung microbiota were analyzed using 16S rRNA deep sequencing and correlated with neutrophil counts in BAL and antibiotic exposure.ResultsOropharyngeal microbial communities clustered separately from lung communities and had higher microbial diversity (p ConclusionsThis study identified a unique microbial profile with altered microbial diversity and metabolic functions in CF airways which is significantly affected by airway inflammation. These results highlight the limitations of using throat swabs as a surrogate to study lower airway microbiome and metagenome in CF children

Illumina paired-end sequencing identifies low frequency T54A+R155K double mutant in mock RNA populations.

<p>RNA transcripts were synthesized from WT, T54A, or T54A+R155K plasmid <i>in vitro</i>. Each mock population of RNA transcripts was constructed according to the proportions indicated in the “Expected” column. The proportion of paired-end reads that harbored T54A single mutant, T54A/R155K double mutant, or WT are shown in the “observed” column (%). While the double mutant at a level below 0.1% was detected (Population 3), it was below our experimentally determined threshold for background error rate of ∼0.2% (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069698#pone-0069698-t002" target="_blank">Table 2</a>).</p

Dynamics of major NS3 variants in liver transplant recipients.

<p>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.</p

Deep sequencing strategy to determine abundance and mutation linkage of NS3 RAVs.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069698#pone.0069698.s001" target="_blank">Supporting Information S1</a>.</p

Mutation linkage analyzed by Illumina paired-end sequencing.

<p>Each row corresponds to an individual sample (control transcripts or clinical samples).</p><p>Each column corresponds to single or double mutant variants associated with PI resistance (WT codons are listed), and their mutation frequency (defined as non-WT) is shown (%). Technical error rate was determined as 0.2% based on the WT control data.</p

Frequency of NS3 resistance-associated variants (RAVs) in (A) chronic HCV and (B) longitudinal liver transplant recipients as determined by Roche/454 pyrosequencing.

<p>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 <i>in vitro</i> 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.</p