Deep Genome Sequencing Research Articles

Estimation of the spontaneous mutation rate per nucleotide site in a Drosophila melanogaster full-sib family.

We employed deep genome sequencing of two parents and 12 of their offspring to estimate the mutation rate per site per generation in a full-sib family of Drosophila melanogaster recently sampled from a natural population. Sites that were homozygous for the same allele in the parents and heterozygous in one or more offspring were categorized as candidate mutations and subjected to detailed analysis. In 1.23 × 10(9) callable sites from 12 individuals, we confirmed six single nucleotide mutations. We estimated the false negative rate in the experiment by generating synthetic mutations using the empirical distributions of numbers of nonreference bases at heterozygous sites in the offspring. The proportion of synthetic mutations at callable sites that we failed to detect was <1%, implying that the false negative rate was extremely low. Our estimate of the point mutation rate is 2.8 × 10(-9) (95% confidence interval = 1.0 × 10(-9) - 6.1 × 10(-9)) per site per generation, which is at the low end of the range of previous estimates, and suggests an effective population size for the species of ∼1.4 × 10(6). At one site, point mutations were present in two individuals, indicating that there had been a premeiotic mutation cluster, although surprisingly one individual had a G→A transition and the other a G→T transversion, possibly associated with error-prone mismatch repair. We also detected three short deletion mutations and no insertions, giving a deletion mutation rate of 1.2 × 10(-9) (95% confidence interval = 0.7 × 10(-9) - 11 × 10(-9)).

Comprehensive Analysis of Long Non-Coding RNAs in Ovarian Cancer Reveals Global Patterns and Targeted DNA Amplification

Long non-coding RNAs (lncRNAs) are emerging as potent regulators of cell physiology, and recent studies highlight their role in tumor development. However, while established protein-coding oncogenes and tumor suppressors often display striking patterns of focal DNA copy-number alteration in tumors, similar evidence is largely lacking for lncRNAs. Here, we report on a genomic analysis of GENCODE lncRNAs in high-grade serous ovarian adenocarcinoma, based on The Cancer Genome Atlas (TCGA) molecular profiles. Using genomic copy-number data and deep coverage transcriptome sequencing, we derived dual copy-number and expression data for 10,419 lncRNAs across 407 primary tumors. We describe global correlations between lncRNA copy-number and expression, and associate established expression subtypes with distinct lncRNA signatures. By examining regions of focal copy-number change that lack protein-coding targets, we identified an intergenic lncRNA on chromosome 1, OVAL, that shows narrow focal genomic amplification in a subset of tumors. While weakly expressed in most tumors, focal amplification coincided with strong OVAL transcriptional activation. Screening of 16 other cancer types revealed similar patterns in serous endometrial carcinomas. This shows that intergenic lncRNAs can be specifically targeted by somatic copy-number amplification, suggestive of functional involvement in tumor initiation or progression. Our analysis provides testable hypotheses and paves the way for further study of lncRNAs based on TCGA and other large-scale cancer genomics datasets.

Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study

SummaryBackgroundSince June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin.MethodsFull genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85–95%, and four 30–50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done.FindingsThree distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction.InterpretationWe present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R0 values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed.FundingSaudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

Comparative analysis of the genome sequences and replication profiles of chikungunya virus isolates within the East, Central and South African (ECSA) lineage.

BackgroundA comparative analysis of the genomic and replication profiles of different geographical chikungunya virus (CHIKV) isolates of the East, Central and South African (ECSA) lineage was performed.FindingsAnalysis of the data revealed the different growth kinetics for the different isolates. Deep genome sequencing analysis further revealed specific amino acid mutations in the viral nsP1, nsP3, nsP4, E1 and E2 proteins in the different isolates. Despite the difference in viral genomic profiles, the virus-induced ultrastructural changes within infected cells remained highly conserved among the different chikungunya virus isolates.ConclusionsThese findings provide insights into the genomic and replication profiles of the re-emerging chikungunya virus isolates of the ECSA lineage.

Neuropilin-1 (Nrp-1) as a prognostic biomarker and potential drug target for pediatric medulloblastoma.

2056 Background: Medulloblastoma survival rates have significantly improved over the last decades due to surgery and chemoradiation regimens. However, pediatric patients with high-risk disease and those with recurrence often succumb to their disease. The majority of children suffer from severe adverse effects of intense therapy regimens. We recently demonstrated the role of the Placental Growth Factor (PlGF)/Nrp-1 signaling pathway in medulloblastoma progression in preclinical models (Snuderl et al., Cell 2013). The aim of this study was to evaluate the role of Nrp-1 as a biomarker and therapeutic target in medulloblastoma patients. Methods: Thirty-two surgical samples of pediatric medulloblastoma were analyzed for expression of Nrp-1 and its ligand PlGF by immunohistochemistry, array-comparative genomic hybridization and deep gene sequencing. On an independent, clinically annotated cohort of 42 medulloblastoma samples, we evaluated the correlation between Nrp-1 expression and 5-year overall survival. The therapeutic relevance of Nrp-1 signaling was tested in a D283 orthotopic human xenograft mouse model using shRNA and a monoclonal antibody against Nrp-1. Results: Nrp-1 expression was detectable in 100% and PlGF in 90% of all medulloblastoma samples. No significant differences in expression could be detected between known histological or molecular medulloblastoma subtypes. Patients with high Nrp-1 expression had a significantly worse 5-year overall survival compared to patients with a low or moderate expression (40% vs. 86%, p = 0.0058). Treatment with an antibody against Nrp-1 or genetic silencing of Nrp-1 with shRNA significantly prolonged survival in a D283 orthotopic human xenograft mouse model (for both experiments: p &lt; 0.01). Conclusions: Nrp-1 is widely expressed across all molecular subgroups of medulloblastoma and its expression levels are inversely associated with survival of patients. Targeting Nrp-1 leads to a significant survival benefit in an orthotopic human xenograft mouse model. This data support the hypothesis that targeting the PlGF/Nrp-1 signaling pathway may be a novel therapy approach for pediatric medulloblastoma.

Full-Genome Deep Sequencing and Phylogenetic Analysis of Novel Human Betacoronavirus

A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient’s sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

Abstract 1131: Novel inhibitor of Notch signaling for the treatment of cancer.

Abstract The Notch pathway is a highly conserved signaling system that plays an important role in development and tissue homeostasis. While Notch mutations are well characterized and implicated in hematological malignancies such as T-cell acute lymphoblastic leukemia, mutations in solid tumors were not reported until recently. With the whole genomic deep sequencing of a large number of samples, deregulated Notch signaling has been implicated in a small percentage of solid tumors such as ovarian, lung, and triple negative breast cancer due to genomic alterations including mutations, amplification, and fusion of Notch pathway components. Inhibition of Notch signaling may provide an attractive targeted cancer therapeutic strategy. We have identified and characterized LY3039478 a novel small molecule that is an exquisitely potent inhibitor of Notch-1 intracellular domain (N1ICD) cleavage with an IC50 of ∼1nM in most of the tumor cell lines tested. We also demonstrate that LY3039478 potently inhibits mutant Notch receptor activity. In a xenograft tumor model, LY3039478 inhibited N1ICD cleavage and expression of Notch-regulated genes in the tumor microenvironment. The inhibition of Notch cleavage also resulted in the induction of apoptosis in a Notch-dependent xenograft model. Using extensive PK/PD data we determined the strength and duration of N1ICD cleavage required for anti-tumor activity which was observed in several xenograft tumors including patient derived tumors representing colon, lung, ovarian, gastric, and breast cancer and glioblastoma. To mitigate the mucoid gasteroentropathy caused by Notch inhibition, PK/PD data were incorporated in devising dosing strategies that identified an optimal intermittent dosing schedule without negatively impacting efficacy. Furthermore, the mucoid gastroentropathy was also mitigated by the prophylactic administration of dexamethasone without negatively impacting the Notch inhibitor mediated efficacy. Mechanistic studies revealed that dexamethasone does not interfere with LY3039478-mediated inhibition of N1ICD cleavage and gene expression but alters the expression of stem cell gene expression in GI tract. In summary, we have characterized an orally bio-available small molecule Notch inhibitor that may provide therapeutic benefit to cancer patients with deregulated Notch signaling. LY3039478 is specifically designed to potently inhibit Notch signaling and is being investigated in Phase I. Citation Format: Mark H. Bender, Hong Gao, Andrew R. Capen, Julia M. Clay, Philip A. Hipskind, Jon K. Reel, Maciej J. Zamek-Gliszczynski, Jason R. Manro, Karim Benhadji, Bharvin K. R. Patel. Novel inhibitor of Notch signaling for the treatment of cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1131. doi:10.1158/1538-7445.AM2013-1131

When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing

ABSTRACTGeobacter sulfurreducens strain KN400 was recovered in previous studies in which a culture of the DL1 strain of G. sulfurreducens served as the inoculum in investigations of microbial current production at low anode potentials (−400 mV versus Ag/AgCl). Differences in the genome sequences of KN400 and DL1 were too great to have arisen from adaptive evolution during growth on the anode. Previous deep sequencing (80-fold coverage) of the DL1 culture failed to detect sequences specific to KN400, suggesting that KN400 was an external contaminant inadvertently introduced into the anode culturing system. In order to evaluate this further, a portion of the gene for OmcS, a c-type cytochrome that both KN400 and DL1 possess, was amplified from the DL1 culture. HiSeq-2000 Illumina sequencing of the PCR product detected the KN400 sequence, which differs from the DL1 sequence at 14 bp, at a frequency of ca. 1 in 105 copies of the DL1 sequence. A similar low frequency of KN400 was detected with quantitative PCR of a KN400-specific gene. KN400 persisted at this frequency after intensive restreaking of isolated colonies from the DL1 culture. However, a culture in which KN400 could no longer be detected was obtained by serial dilution to extinction in liquid medium. The KN400-free culture could not grow on an anode poised at −400 mV. Thus, KN400 cryptically persisted in the culture dominated by DL1 for more than a decade, undetected by even deep whole-genome sequencing, and was only fortuitously uncovered by the unnatural selection pressure of growth on a low-potential electrode.

Gene Expression ‘Signature‘ Limitations and Genome Architecture-Based Perspectives for Robust Cancer Biomarkers

Everybody agrees: patients, healthcare systems and society urgently need cancer biomarkers. However, the way to reach this goal is debated. Predictive medicine to tailor the specific optimal treatment in specific subgroups or individual patients has been one of the most intensive areas of research attracting major funding and producing countless articles, but clinical progress is modest. In this article, emphasis is given on understanding the challenges of personalized medicine, the pitfalls of simplif ied omics studies and the opportunities to achieve, at last, clinical success with emerging deep-genome sequencing and a functional comprehensive understanding of complex dynamic interacting biological systems. This deeper insight into gene expression and cellular signaling transduction networks rationally promises the discovery of robust biomarkers for clinical use. Over recent decades, traditional tumor node metastasis staging, clinicopathologic factors and single-gene abnormalities have continued to remain the standard classification for therapeutic decisions. However, evidence implies that the multigenic model for tumorigenesis and metastasis is involved for the vast majority of cancer patients. High-throughput (HT) array-based technologies have been developed over the last 15 years to provide the capacity of profiling measurement of multiple differentially expressed genes simultaneously. This capacity has yielded a simplified notion that applying microarrays in cancer tissue samples from individual patients could produce new diagnostics, and prognostic and predictive expression prof iling-based tools. This research boom has been associated with multiple multigene assays termed gene ‘signatures’. Some of these signatures have been commercialized and have become available.

Identification of differentially methylated regions using streptavidin bisulfite ligand methylation enrichment (SuBLiME), a new method to enrich for methylated DNA prior to deep bisulfite genomic sequencing

We have developed a method that enriches for methylated cytosines by capturing the fraction of bisulfite-treated DNA with unconverted cytosines. The method, called streptavidin bisulfite ligand methylation enrichment (SuBLiME), involves the specific labeling (using a biotin-labeled nucleotide ligand) of methylated cytosines in bisulfite-converted DNA. This step is then followed by affinity capture, using streptavidin-coupled magnetic beads. SuBLiME is highly adaptable and can be combined with deep sequencing library generation and/or genomic complexity-reduction. In this pilot study, we enriched methylated DNA from Csp6I-cut complexity-reduced genomes of colorectal cancer cell lines (HCT-116, HT-29 and SW-480) and normal blood leukocytes with the aim of discovering colorectal cancer biomarkers. Enriched libraries were sequenced with SOLiD-3 technology. In pairwise comparisons, we scored a total of 1,769 gene loci and 33 miRNA loci as differentially methylated between the cell lines and leukocytes. Of these, 516 loci were differently methylated in at least two promoter-proximal CpG sites over two discrete Csp6I fragments. Identified methylated gene loci were associated with anatomical development, differentiation and cell signaling. The data correlated with good agreement to a number of published colorectal cancer DNA methylation biomarkers and genomic data sets. SuBLiME is effective in the enrichment of methylated nucleic acid and in the detection of known and novel biomarkers.

Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines

Forward genetic screens have identified numerous genes involved in development and metabolism, and remain a cornerstone of biological research. However, to locate a causal mutation, the practice of crossing to a polymorphic background to generate a mapping population can be problematic if the mutant phenotype is difficult to recognize in the hybrid F2 progeny, or dependent on parental specific traits. Here in a screen for leaf hyponasty mutants, we have performed a single backcross of an Ethane Methyl Sulphonate (EMS) generated hyponastic mutant to its parent. Whole genome deep sequencing of a bulked homozygous F2 population and analysis via the Next Generation EMS mutation mapping pipeline (NGM) unambiguously determined the causal mutation to be a single nucleotide polymorphisim (SNP) residing in HASTY, a previously characterized gene involved in microRNA biogenesis. We have evaluated the feasibility of this backcross approach using three additional SNP mapping pipelines; SHOREmap, the GATK pipeline, and the samtools pipeline. Although there was variance in the identification of EMS SNPs, all returned the same outcome in clearly identifying the causal mutation in HASTY. The simplicity of performing a single parental backcross and genome sequencing a small pool of segregating mutants has great promise for identifying mutations that may be difficult to map using conventional approaches.

Composite Single Cell Genetics and Clonal Phylogeny in Acute Lymphoblastic Leukaemia

AbstractAbstract 122Cancer clone development is widely regarded as an evolutionary or Darwinian process of genetic diversification and natural (or therapeutic) selection within tissue ecosystems. Emerging studies are providing strong evidence that dynamic and complex branching sub-clonal genetic architectures are a common feature of cancer (Greaves M and Maley CC Nature 2012). This complexity may underpin the intransigence of advanced cancer to therapeutic control, particularly as the critical ’driver’ cells – cancer or leukaemic stem cells, also appear to be genetically diverse within individual patients (Anderson K et al Nature 2011, Notta F et al Nature 2011). Sub-clonal architecture can only be fully determined through the study of large numbers of single cells uniformly sampled from the individual cancer of interest and assessed for composite genotype. Various technologies and approaches from fluorescent in situ hybridisation (FISH) to whole-genome sequencing of single cells have been applied to cancer and leukaemic cells but each approach has limitations. We have developed a novel multiplex microfluidic Q-PCR approach that allows unbiased single cell sampling, high throughput analysis of hundreds of individual cells and simultaneous detection of multiple genetic alterations in a single cell, including fusion genes, DNA copy number alterations (CNAs) and sequence-based mutations.As a proof of principle study we have applied this technique to REH, an acute lymphoblastic leukaemia (ALL) cell line that harbors the ETV6-RUNX1 fusion and a SNP in the EPO receptor gene, which we used as a surrogate mutation. We further determined a detailed sub-clonal genetic architecture for two ETV6-RUNX1 positive ALL patient samples with multiple point mutations and copy number alterations (determined by whole-genome sequencing) by interrogating approximately 400 flow cytometry sorted single cells with validation by FISH and standard sequencing. Briefly, single cells were lysed prior to multiplex specific (DNA) target amplification (STA) and Q-PCR using the 96.96 dynamic microfluidic array and the BioMarkï HD (Fluidigm, UK). Phylogenetic trees were constructed using maximum parsimony with PAUP analysis software.Interrogation of REH revealed that all single cells registered the ETV6-RUNX1 fusion and EPO receptor SNP, but 42% of cells gained either 1 or 2 additional copies of chromosome 21. Patient sample data revealed branching sub-clonal architectures in Case A in which all leukaemic cells harbored the fusion with additional point mutations but only sub-clones showed CNAs. In contrast, the sub-clonal architecture of Case B showed that whilst the ETV6-RUNX1 fusion was the earliest (or universal) genomic event, CNAs were relatively early events preceding the acquisition of point mutations (Figure 1). In both cases, the numerically predominant sub-clone harbored both point mutations and CNAs in addition to the presumptive initiating lesion, ETV6-RUNX1.These detailed and complex sub-clonal architectures would be masked by other genetic techniques. Single cell genetics coupled with deep genome sequencing is now technically feasible and provides an accurate portrait of the dynamic clonal complexity in leukaemia (and other cancers). Variegated genetics and clonal complexity in individual leukaemias has important implications for our understanding of molecular pathogenesis and for therapeutic targeting. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A deeper view of transmitted/founder viruses using 454 whole genome deep sequencing

Background Understanding the composition of transmitted/founder viruses that represent the primary target for vaccineelicited responses is highly relevant to the design of an effective HIV-1 vaccine. The identification of transmitted and early founder viruses in primary infection has remained remarkably opaque, but recent advances in sequencing technologies have enabled a more comprehensive and sensitive assessment of the properties of transmitted/founder viruses.

GENOMIC BASIS OF AGING AND LIFE-HISTORY EVOLUTION INDROSOPHILA MELANOGASTER

Natural diversity in aging and other life-history patterns is a hallmark of organismal variation. Related species, populations, and individuals within populations show genetically based variation in life span and other aspects of age-related performance. Population differences are especially informative because these differences can be large relative to within-population variation and because they occur in organisms with otherwise similar genomes. We used experimental evolution to produce populations divergent for life span and late-age fertility and then used deep genome sequencing to detect sequence variants with nucleotide-level resolution. Several genes and genome regions showed strong signatures of selection, and the same regions were implicated in independent comparisons, suggesting that the same alleles were selected in replicate lines. Genes related to oogenesis, immunity, and protein degradation were implicated as important modifiers of late-life performance. Expression profiling and functional annotation narrowed the list of strong candidate genes to 38, most of which are novel candidates for regulating aging. Life span and early age fecundity were negatively correlated among populations; therefore, the alleles we identified also are candidate regulators of a major life-history trade-off. More generally, we argue that hitchhiking mapping can be a powerful tool for uncovering the molecular bases of quantitative genetic variation.

Abstract LB-453: Phase II trial of afatinib, with or without vinorelbine (V), for the treatment of HER2-overexpressing inflammatory breast cancer (IBC). A biomarker driven study using ultra deep genome sequencing technology

Abstract Background:Personalized medicine in breast cancer (BC), by improving pt selection and predicting drug response, is challenging. Acquisition of tumor multidrug resistance is inevitable in advanced BC. Drug resistance is highly complex and single mechanisms fail to explain this phenomenon. Ultra-deep whole genome sequencing has shown unexpected and profound complexity and intra/inter-tumor heterogeneity. Strategies to identify biomarkers and drug resistance mechanisms will likely require in-depth analysis of pre/post-treatment resistant tumor biopsies to study dynamic changes in clonal tumor architecture during therapy. IBC is a rare, aggressive form of BC. It commonly lacks ER/PR, and more frequently harbors HER2 gene amplification and EGFR overexpression. Afatinib is an irreversible ErbB Family Blocker with preclinical activity in trastuzumab (T)-resistant cell lines, HER2+ tumor xenografts and HER2- SUM 149 xenograft model, derived from an IBC cell line. Its clinical efficacy was shown in T-resistant, HER2+ metastatic BC. This biomarker-driven study in pts with T pretreated/naïve, HER2+ IBC, investigates efficacy/safety of afatinib with/without V, upon disease progression (PD) on afatinib monotherapy, and changes in clonal tumor architecture that occur through treatment.Methods: Pts with HER2+ IBC are recruited in 2 parallel cohorts of 20 pts each: T-naïve and T failure. All pts start afatinib monotherapy (40 mg/d oral; Part A). Upon PD, pts may receive afatinib (40 mg/d) + V (IV 25 mg/m2/week; Part B). Study endpoints:clinical benefit rate (CR, PR or SD) for ≥6 months, ORR, PFS, OS and safety. In Part A fresh tumor biopsies are taken pretreatment + upon PD and blood samples are taken pretreatment for ultra-deep whole genome sequencing (via DNA/RNA analysis) including gene expression, copy number and proteomic analysis to explore predictive markers of response/resistance to afatinib, and to describe IBC population which may benefit most. The use of pre/post-treatment ultra-deep whole genome sequencing aims to: identify bottlenecking resistance mechanisms to ErbB Family Blockers, identify pretreatment subclonal mutations that predict outcome to afatinib, establish trials network and tissue collection methodology, develop greater understanding of IBC biology and clarify the sensitivity of HER2+ IBC to HER2 inhibitors.Conclusions: Understanding subclonal architecture will be key in advancing our knowledge of resistance mechanisms. Evolutionary bottlenecking during drug resistance offers a tractable process to predict drug outcome and identify novel targets. This is the first prospective clinical trial to integrate whole genome sequencing into trial design to assess proof of principle of this personalized cancer medicine approach to improve pt stratification for therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-453. doi:1538-7445.AM2012-LB-453

Whole Genome Deep Sequencing of HIV-1 Reveals the Impact of Early Minor Variants Upon Immune Recognition During Acute Infection

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

Huvariome: a web server resource of whole genome next-generation sequencing allelic frequencies to aid in pathological candidate gene selection

BackgroundNext generation sequencing provides clinical research scientists with direct read out of innumerable variants, including personal, pathological and common benign variants. The aim of resequencing studies is to determine the candidate pathogenic variants from individual genomes, or from family-based or tumor/normal genome comparisons. Whilst the use of appropriate controls within the experimental design will minimize the number of false positive variations selected, this number can be reduced further with the use of high quality whole genome reference data to minimize false positives variants prior to candidate gene selection. In addition the use of platform related sequencing error models can help in the recovery of ambiguous genotypes from lower coverage data.DescriptionWe have developed a whole genome database of human genetic variations, Huvariome, determined by whole genome deep sequencing data with high coverage and low error rates. The database was designed to be sequencing technology independent but is currently populated with 165 individual whole genomes consisting of small pedigrees and matched tumor/normal samples sequenced with the Complete Genomics sequencing platform. Common variants have been determined for a Benelux population cohort and represented as genotypes alongside the results of two sets of control data (73 of the 165 genomes), Huvariome Core which comprises 31 healthy individuals from the Benelux region, and Diversity Panel consisting of 46 healthy individuals representing 10 different populations and 21 samples in three Pedigrees. Users can query the database by gene or position via a web interface and the results are displayed as the frequency of the variations as detected in the datasets. We demonstrate that Huvariome can provide accurate reference allele frequencies to disambiguate sequencing inconsistencies produced in resequencing experiments. Huvariome has been used to support the selection of candidate cardiomyopathy related genes which have a homozygous genotype in the reference cohorts. This database allows the users to see which selected variants are common variants (> 5% minor allele frequency) in the Huvariome core samples, thus aiding in the selection of potentially pathogenic variants by filtering out common variants that are not listed in one of the other public genomic variation databases. The no-call rate and the accuracy of allele calling in Huvariome provides the user with the possibility of identifying platform dependent errors associated with specific regions of the human genome.ConclusionHuvariome is a simple to use resource for validation of resequencing results obtained by NGS experiments. The high sequence coverage and low error rates provide scientists with the ability to remove false positive results from pedigree studies. Results are returned via a web interface that displays location-based genetic variation frequency, impact on protein function, association with known genetic variations and a quality score of the variation base derived from Huvariome Core and the Diversity Panel data. These results may be used to identify and prioritize rare variants that, for example, might be disease relevant. In testing the accuracy of the Huvariome database, alleles of a selection of ambiguously called coding single nucleotide variants were successfully predicted in all cases. Data protection of individuals is ensured by restricted access to patient derived genomes from the host institution which is relevant for future molecular diagnostics.

Whole genome deep sequencing from single cells for preimplantation genetic diagnosis

FISH and PCR are two main methodologies being used for preimplantation genetic diagnosis (PGD) in the last two decades. Recent advancements in micro-array technology expand the applications of PGD, particularly for preimplatation genetic screening (PGS) to 24 chromosome analysis. A much more powerful approach, next generation sequencing (NGS), is emerging and may have the potential to replace current methodologies for comprehensive genetic analysis. The purpose of this study was to prove the concept that NGS for whole genome analysis on the amplified products from single cells can be used for chromosome copy number assessment and single gene disorder detection. Proof of concept. The whole genome of single cells from known fibroblast cell lines was amplified using commercially available kits. Libraries from amplified DNA were prepared and shotgun deep sequencing was performed using the Roche 454 (Junior) platform. Reads were assembled and aligned using a publically available sequence (Nature, 2008; 456:60) and were analyzed with BWA-SW algorithm. Copy number variation (CNV) was analyzed with MATLAB SegSeq software. NGS on whole genome amplified products from single cells successfully detected the chromosome permutations in cell lines with chromosomal abnormalities. In the experiments we performed, a single NGS run could generate more than one hundred thousand reads, with an average length of 363bp. Computational analysis of the reads indicated the known genetic mutations could be detected. This work indicates that next generation deep sequencing provides high sensitivity and coverage for single cell comprehensive genetic analysis. Thus proves unequivocally that NGS will be a viable technique for clinical PGD in the near future. The method's limitations that exist today, such as speed and cost, are expected to be eliminated in the coming years.

Whole Genome Sequencing and Complete Genetic Analysis Reveals Novel Pathways to Glycopeptide Resistance in Staphylococcus aureus

The precise mechanisms leading to the emergence of low-level glycopeptide resistance in Staphylococcus aureus are poorly understood. In this study, we used whole genome deep sequencing to detect differences between two isogenic strains: a parental strain and a stable derivative selected stepwise for survival on 4 µg/ml teicoplanin, but which grows at higher drug concentrations (MIC 8 µg/ml). We uncovered only three single nucleotide changes in the selected strain. Nonsense mutations occurred in stp1, encoding a serine/threonine phosphatase, and in yjbH, encoding a post-transcriptional negative regulator of the redox/thiol stress sensor and global transcriptional regulator, Spx. A missense mutation (G45R) occurred in the histidine kinase sensor of cell wall stress, VraS. Using genetic methods, all single, pairwise combinations, and a fully reconstructed triple mutant were evaluated for their contribution to low-level glycopeptide resistance. We found a synergistic cooperation between dual phospho-signalling systems and a subtle contribution from YjbH, suggesting the activation of oxidative stress defences via Spx. To our knowledge, this is the first genetic demonstration of multiple sensor and stress pathways contributing simultaneously to glycopeptide resistance development. The multifactorial nature of glycopeptide resistance in this strain suggests a complex reprogramming of cell physiology to survive in the face of drug challenge.

Hypervirulent Chlamydia trachomatis Clinical Strain Is a Recombinant between Lymphogranuloma Venereum (L 2 ) and D Lineages

ABSTRACTChlamydia trachomatis is an obligate intracellular bacterium that causes a diversity of severe and debilitating diseases worldwide. Sporadic and ongoing outbreaks of lymphogranuloma venereum (LGV) strains among men who have sex with men (MSM) support the need for research on virulence factors associated with these organisms. Previous analyses have been limited to single genes or genomes of laboratory-adapted reference strain L2/434 and outbreak strain L2b/UCH-1/proctitis. We characterized an unusual LGV strain, termed L2c, isolated from an MSM with severe hemorrhagic proctitis. L2c developed nonfusing, grape-like inclusions and a cytotoxic phenotype in culture, unlike the LGV strains described to date. Deep genome sequencing revealed that L2c was a recombinant of L2 and D strains with conserved clustered regions of genetic exchange, including a 78-kb region and a partial, yet functional, toxin gene that was lost with prolonged culture. Indels (insertions/deletions) were discovered in an ftsK gene promoter and in the tarp and hctB genes, which encode key proteins involved in replication, inclusion formation, and histone H1-like protein activity, respectively. Analyses suggest that these indels affect gene and/or protein function, supporting the in vitro and disease phenotypes. While recombination has been known to occur for C. trachomatis based on gene sequence analyses, we provide the first whole-genome evidence for recombination between a virulent, invasive LGV strain and a noninvasive common urogenital strain. Given the lack of a genetic system for producing stable C. trachomatis mutants, identifying naturally occurring recombinants can clarify gene function and provide opportunities for discovering avenues for genomic manipulation.