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Auto Tag: Circular consensus sequencing

July 19, 2019

Single-molecule sequencing reveals complex genomic variation of hepatitis B virus during 15 years of chronic infection following liver transplantation.

Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV) replicates via an RNA intermediate and is error-prone, leading to rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome, generated via splicing (spHBV). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. SpHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from longitudinal samples of a post-liver transplant CHB subject, collected over a 15-year period that included the liver explant. By employing novel bioinformatics methods, this analysis showed a complex dynamics of the viral population across a period of changing treatment regimens. The spHBV detected in the liver explant remained present post-transplantation, along with emergence of a highly diverse novel spHBV population as well as variants with multiple deletions in the preS genes. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occur with known drug resistant mutations, highlight the relevance of using full genome deep sequencing and support the hypothesis that drug resistance involves interactions across the full-length HBV genome.Single-molecule sequencing allowed characterising, in unprecedented detail, the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimes. This analysis also showed rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open source bioinformatics tools are freely available, with the capacity to easily identify potential spliced variants from deep sequencing data. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 19, 2019

A comparison of tools for the simulation of genomic next-generation sequencing data.

Computer simulation of genomic data has become increasingly popular for assessing and validating biological models or for gaining an understanding of specific data sets. Several computational tools for the simulation of next-generation sequencing (NGS) data have been developed in recent years, which could be used to compare existing and new NGS analytical pipelines. Here we review 23 of these tools, highlighting their distinct functionality, requirements and potential applications. We also provide a decision tree for the informed selection of an appropriate NGS simulation tool for the specific question at hand.

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July 19, 2019

Rapid sequencing of complete env genes from primary HIV-1 samples

The ability to study rapidly evolving viral populations has been constrained by the read length of next-generation sequencing approaches and the sampling depth of single-genome amplification methods. Here, we develop and characterize a method using Pacific Biosciences Single Molecule, Real-Time (SMRT) sequencing technology to sequence multiple, intact full-length human immunodeficiency virus-1 env genes amplified from viral RNA populations circulating in blood, and provide computational tools for analyzing and visualizing these data.

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July 19, 2019

Variation and evolution in the glutamine-rich repeat region of Drosophila argonaute-2.

RNA interference pathways mediate biological processes through Argonaute-family proteins, which bind small RNAs as guides to silence complementary target nucleic acids . In insects and crustaceans Argonaute-2 silences viral nucleic acids, and therefore acts as a primary effector of innate antiviral immunity. Although the function of the major Argonaute-2 domains, which are conserved across most Argonaute-family proteins, are known, many invertebrate Argonaute-2 homologs contain a glutamine-rich repeat (GRR) region of unknown function at the N-terminus . Here we combine long-read amplicon sequencing of Drosophila Genetic Reference Panel (DGRP) lines with publicly available sequence data from many insect species to show that this region evolves extremely rapidly and is hyper-variable within species. We identify distinct GRR haplotype groups in Drosophila melanogaster, and suggest that one of these haplotype groups has recently risen to high frequency in a North American population. Finally, we use published data from genome-wide association studies of viral resistance in D. melanogaster to test whether GRR haplotypes are associated with survival after virus challenge. We find a marginally significant association with survival after challenge with Drosophila C Virus in the DGRP, but we were unable to replicate this finding using lines from the Drosophila Synthetic Population Resource panel. Copyright © 2016 Palmer and Obbard.

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July 19, 2019

High frequency of mitochondrial DNA mutations in HIV-infected treatment-experienced individuals.

We recently observed a decrease in deoxyribonucleotide (dNTP) pools in HIV-infected individuals on antiretroviral therapy (ART). Alterations in dNTPs result in mutations in mitochondrial DNA (mtDNA) in cell culture and animal models. Therefore, we investigated whether ART is associated with mitochondrial genome sequence variation in peripheral blood mononuclear cells (PBMCs) of HIV-infected treatment-experienced individuals.In this substudy of a case-control study, 71 participants were included: 22 ‘cases’, who were HIV-infected treatment-experienced patients with mitochondrial toxicity, 25 HIV-infected treatment-experienced patients without mitochondrial toxicity, and 24 HIV-uninfected controls. Total DNA was extracted from PBMCs and purified polymerase chain reaction (PCR) products were subjected to third-generation sequencing using the PacBio Single Molecule Real-Time (SMRT) sequencing technology. The sequences were aligned against the revised Cambridge reference sequence for human mitochondrial DNA (NC_012920.1) for detection of variants.We identified a total of 123 novel variants, 39 of them in the coding region. HIV-infected treatment-experienced patients with and without toxicity had significantly higher average numbers of mitochondrial variants per participant than HIV-uninfected controls. We observed a higher burden of mtDNA large-scale deletions in HIV-infected treatment-experienced patients with toxicity compared with HIV-uninfected controls (P = 0.02). The frequency of mtDNA molecules containing a common deletion (mt.d4977) was higher in HIV-infected treatment-experienced patients with toxicity compared with HIV-uninfected controls (P = 0.06). There was no statistically significant difference in mtDNA variants between HIV-infected treatment-experienced patients with and without toxicity.The frequency of mtDNA variants (mutations and large-scale deletions) was higher in HIV-infected treatment-experienced patients with or without ART-induced toxicity than in uninfected controls.© 2016 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association.

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July 19, 2019

High throughput random mutagenesis and Single Molecule Real Time Sequencing of the muscle nicotinic acetylcholine receptor.

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the a1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each a1 mutant was co-transfected with wildtype ß1, d, and e subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either a-bungarotoxin or tubocurarine. Eight a1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

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July 19, 2019

Comparative DNA methylation and gene expression analysis identifies novel genes for structural congenital heart diseases.

For the majority of congenital heart diseases (CHDs), the full complexity of the causative molecular network, which is driven by genetic, epigenetic, and environmental factors, is yet to be elucidated. Epigenetic alterations are suggested to play a pivotal role in modulating the phenotypic expression of CHDs and their clinical course during life. Candidate approaches implied that DNA methylation might have a developmental role in CHD and contributes to the long-term progress of non-structural cardiac diseases. The aim of the present study is to define the postnatal epigenome of two common cardiac malformations, representing epigenetic memory, and adaption to hemodynamic alterations, which are jointly relevant for the disease course.We present the first analysis of genome-wide DNA methylation data obtained from myocardial biopsies of Tetralogy of Fallot (TOF) and ventricular septal defect patients. We defined stringent sets of differentially methylated regions between patients and controls, which are significantly enriched for genomic features like promoters, exons, and cardiac enhancers. For TOF, we linked DNA methylation with genome-wide expression data and found a significant overlap for hypermethylated promoters and down-regulated genes, and vice versa. We validated and replicated the methylation of selected CpGs and performed functional assays. We identified a hypermethylated novel developmental CpG island in the promoter of SCO2 and demonstrate its functional impact. Moreover, we discovered methylation changes co-localized with novel, differential splicing events among sarcomeric genes as well as transcription factor binding sites. Finally, we demonstrated the interaction of differentially methylated and expressed genes in TOF with mutated CHD genes in a molecular network.By interrogating DNA methylation and gene expression data, we identify two novel mechanism contributing to the phenotypic expression of CHDs: aberrant methylation of promoter CpG islands and methylation alterations leading to differential splicing. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

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July 19, 2019

Single-molecule sequencing revealing the presence of distinct JC polyomavirus populations in patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a fatal disease caused by reactivation of JC polyomavirus (JCPyV) in immunosuppressed individuals and lytic infection by neurotropic JCPyV in glial cells. The exact content of neurotropic mutations within individual JCPyV strains has not been studied to our knowledge.We exploited the capacity of single-molecule real-time sequencing technology to determine the sequence of complete JCPyV genomes in single reads. The method was used to precisely characterize individual neurotropic JCPyV strains of 3 patients with PML without the bias caused by assembly of short sequence reads.In the cerebrospinal fluid sample of a 73-year-old woman with rapid PML onset, 3 distinct JCPyV populations could be identified. All viral populations were characterized by rearrangements within the noncoding regulatory region (NCCR) and 1 point mutation, S267L in the VP1 gene, suggestive of neurotropic strains. One patient with PML had a single neurotropic strain with rearranged NCCR, and 1 patient had a single strain with small NCCR alterations.We report here, for the first time, full characterization of individual neurotropic JCPyV strains in the cerebrospinal fluid of patients with PML. It remains to be established whether PML pathogenesis is driven by one or several neurotropic strains in an individual.

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July 19, 2019

Examining sources of error in PCR by single-molecule sequencing.

Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification.

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July 19, 2019

Antibody 10-1074 suppresses viremia in HIV-1-infected individuals.

Monoclonal antibody 10-1074 targets the V3 glycan supersite on the HIV-1 envelope (Env) protein. It is among the most potent anti-HIV-1 neutralizing antibodies isolated so far. Here we report on its safety and activity in 33 individuals who received a single intravenous infusion of the antibody. 10-1074 was well tolerated and had a half-life of 24.0 d in participants without HIV-1 infection and 12.8 d in individuals with HIV-1 infection. Thirteen individuals with viremia received the highest dose of 30 mg/kg 10-1074. Eleven of these participants were 10-1074-sensitive and showed a rapid decline in viremia by a mean of 1.52 log10 copies/ml. Virologic analysis revealed the emergence of multiple independent 10-1074-resistant viruses in the first weeks after infusion. Emerging escape variants were generally resistant to the related V3-specific antibody PGT121, but remained sensitive to antibodies targeting nonoverlapping epitopes, such as the anti-CD4-binding-site antibodies 3BNC117 and VRC01. The results demonstrate the safety and activity of 10-1074 in humans and support the idea that antibodies targeting the V3 glycan supersite might be useful for the treatment and prevention of HIV-1 infection.

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July 19, 2019

Characterization of hepatitis C virus (HCV) envelope diversification from acute to chronic infection within a sexually transmitted HCV cluster by using single-molecule, real-time sequencing.

In contrast to other available next-generation sequencing platforms, PacBio single-molecule, real-time (SMRT) sequencing has the advantage of generating long reads albeit with a relatively higher error rate in unprocessed data. Using this platform, we longitudinally sampled and sequenced the hepatitis C virus (HCV) envelope genome region (1,680 nucleotides [nt]) from individuals belonging to a cluster of sexually transmitted cases. All five subjects were coinfected with HIV-1 and a closely related strain of HCV genotype 4d. In total, 50 samples were analyzed by using SMRT sequencing. By using 7 passes of circular consensus sequencing, the error rate was reduced to 0.37%, and the median number of sequences was 612 per sample. A further reduction of insertions was achieved by alignment against a sample-specific reference sequence. However, in vitro recombination during PCR amplification could not be excluded. Phylogenetic analysis supported close relationships among HCV sequences from the four male subjects and subsequent transmission from one subject to his female partner. Transmission was characterized by a strong genetic bottleneck. Viral genetic diversity was low during acute infection and increased upon progression to chronicity but subsequently fluctuated during chronic infection, caused by the alternate detection of distinct coexisting lineages. SMRT sequencing combines long reads with sufficient depth for many phylogenetic analyses and can therefore provide insights into within-host HCV evolutionary dynamics without the need for haplotype reconstruction using statistical algorithms.IMPORTANCE Next-generation sequencing has revolutionized the study of genetically variable RNA virus populations, but for phylogenetic and evolutionary analyses, longer sequences than those generated by most available platforms, while minimizing the intrinsic error rate, are desired. Here, we demonstrate for the first time that PacBio SMRT sequencing technology can be used to generate full-length HCV envelope sequences at the single-molecule level, providing a data set with large sequencing depth for the characterization of intrahost viral dynamics. The selection of consensus reads derived from at least 7 full circular consensus sequencing rounds significantly reduced the intrinsic high error rate of this method. We used this method to genetically characterize a unique transmission cluster of sexually transmitted HCV infections, providing insight into the distinct evolutionary pathways in each patient over time and identifying the transmission-associated genetic bottleneck as well as fluctuations in viral genetic diversity over time, accompanied by dynamic shifts in viral subpopulations. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation.

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either PacBio or Oxford Nanopore technologies, and achieves a contig NG50 of greater than 21 Mbp on both human and Drosophila melanogaster PacBio datasets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes. Published by Cold Spring Harbor Laboratory Press.

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July 19, 2019

Evolution of multi-drug resistant HCV clones from pre-existing resistant-associated variants during direct-acting antiviral therapy determined by third-generation sequencing.

Resistance-associated variant (RAV) is one of the most significant clinical challenges in treating HCV-infected patients with direct-acting antivirals (DAAs). We investigated the viral dynamics in patients receiving DAAs using third-generation sequencing technology. Among 283 patients with genotype-1b HCV receiving daclatasvir?+?asunaprevir (DCV/ASV), 32 (11.3%) failed to achieve sustained virological response (SVR). Conventional ultra-deep sequencing of HCV genome was performed in 104 patients (32 non-SVR, 72 SVR), and detected representative RAVs in all non-SVR patients at baseline, including Y93H in 28 (87.5%). Long contiguous sequences spanning NS3 to NS5A regions of each viral clone in 12 sera from 6 representative non-SVR patients were determined by third-generation sequencing, and showed the concurrent presence of several synonymous mutations linked to resistance-associated substitutions in a subpopulation of pre-existing RAVs and dominant isolates at treatment failure. Phylogenetic analyses revealed close genetic distances between pre-existing RAVs and dominant RAVs at treatment failure. In addition, multiple drug-resistant mutations developed on pre-existing RAVs after DCV/ASV in all non-SVR cases. In conclusion, multi-drug resistant viral clones at treatment failure certainly originated from a subpopulation of pre-existing RAVs in HCV-infected patients. Those RAVs were selected for and became dominant with the acquisition of multiple resistance-associated substitutions under DAA treatment pressure.

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July 19, 2019

Quasispecies composition and evolution of a typical Zika virus clinical isolate from Suriname.

The arthropod-borne Zika virus (ZIKV) is currently causing a major international public health threat in the Americas. This study describes the isolation of ZIKV from the plasma of a 29-year-old female traveler that developed typical symptoms, like rash, fever and headache upon return from Suriname. The complete genome sequence including the 5′ and 3′ untranslated regions was determined and phylogenetic analysis showed the isolate clustering within the Asian lineage, close to other viruses that have recently been isolated in the Americas. In addition, the viral quasispecies composition was analyzed by single molecule real time sequencing, which suggested a mutation frequency of 1.4?×?10(-4) for this ZIKV isolate. Continued passaging of the virus in cell culture led to the selection of variants with mutations in NS1 and the E protein. The latter might influence virus binding to cell surface heparan sulfate.

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July 19, 2019

Quality control of the traditional patent medicine Yimu Wan based on SMRT Sequencing and DNA barcoding.

Substandard traditional patent medicines may lead to global safety-related issues. Protecting consumers from the health risks associated with the integrity and authenticity of herbal preparations is of great concern. Of particular concern is quality control for traditional patent medicines. Here, we establish an effective approach for verifying the biological composition of traditional patent medicines based on single-molecule real-time (SMRT) sequencing and DNA barcoding. Yimu Wan (YMW), a classical herbal prescription recorded in the Chinese Pharmacopoeia, was chosen to test the method. Two reference YMW samples were used to establish a standard method for analysis, which was then applied to three different batches of commercial YMW samples. A total of 3703 and 4810 circular-consensus sequencing (CCS) reads from two reference and three commercial YMW samples were mapped to the ITS2 and psbA-trnH regions, respectively. Moreover, comparison of intraspecific genetic distances based on SMRT sequencing data with reference data from Sanger sequencing revealed an ITS2 and psbA-trnH intergenic spacer that exhibited high intraspecific divergence, with the sites of variation showing significant differences within species. Using the CCS strategy for SMRT sequencing analysis was adequate to guarantee the accuracy of identification. This study demonstrates the application of SMRT sequencing to detect the biological ingredients of herbal preparations. SMRT sequencing provides an affordable way to monitor the legality and safety of traditional patent medicines.

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