Tracking the evolution of dengue virus strains D2S10 and D2S20 by 454 pyrosequencing.
Ontology highlight
ABSTRACT:
SUBMITTER: Makhluf H
PROVIDER: S-EPMC3544829 | biostudies-literature | 2013
REPOSITORIES: biostudies-literature
Publications
Tracking the evolution of dengue virus strains D2S10 and D2S20 by 454 pyrosequencing.
Makhluf Huda H Buck Michael D MD King Kevin K Perry Stuart T ST Henn Matthew R MR Shresta Sujan S
PloS one 20130114 1
Dengue virus is the most prevalent mosquito-borne virus worldwide. In this study, we used pyrosequencing to analyze the whole viral genome of two mouse-adapted strains, D2S10 and D2S20, that induce a dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)-like lethal disease in mice lacking the type I and/or type II interferon receptors. Previous experiments with D2S10 indicated that N124D and K128E mutations in the envelope protein were responsible for the severe disease induced in mice compar ...[more]
Similar Datasets
Project description:Adaptation of avian influenza viruses (AIVs) from waterfowl to domestic poultry with a deletion in the neuraminidase (NA) stalk has already been reported. The way the virus undergoes this evolution, however, is thus far unclear. We address this question using pyrosequencing of duck and turkey low-pathogenicity AIVs. Ducks and turkeys were sampled at the very beginning of an H6N1 outbreak, and turkeys were swabbed again 8 days later. NA stalk deletions were evidenced in turkeys by Sanger sequencing. To further investigate viral evolution, 454 pyrosequencing was performed: for each set of samples, up to 41,500 reads of ca. 400 bp were generated and aligned. Genetic polymorphisms between duck and turkey viruses were tracked on the whole genome. NA deletion was detected in less than 2% of reads in duck feces but in 100% of reads in turkey tracheal specimens collected at the same time. Further variations in length were observed in NA from turkeys 8 days later. Similarly, minority mutants emerged on the hemagglutinin (HA) gene, with substitutions mostly in the receptor binding site on the globular head. These critical changes suggest a strong evolutionary pressure in turkeys. The increasing performances of next-generation sequencing technologies should enable us to monitor the genomic diversity of avian influenza viruses and early emergence of potentially pathogenic variants within bird flocks. The present study, based on 454 pyrosequencing, suggests that NA deletion, an example of AIV adaptation from waterfowl to domestic poultry, occurs by selection rather than de novo emergence of viral mutants.
| S-EPMC3421805 | biostudies-literature
Project description:Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and extent of genetic diversity in the envelope (E) gene of DENV serotype 1 representing different lineages histories. A region of the DENV genome spanning the E gene was amplified and sequenced by Roche/454 pyrosequencing. The pyrosequencing results identified distinct sub-populations (haplotypes) for each DENV-1 E gene. A phylogenetic tree was constructed with the consensus DENV-1 E gene nucleotide sequences, and the sequences of each constructed haplotype showed that the haplotypes segregated with the Sanger consensus sequence of the population from which they were drawn. Haplotypes determined through pyrosequencing identified a recombinant DENV genome that could not be identified through Sanger sequencing. Nucleotide level sequence diversities of DENV-1 populations determined from SNP analysis were very low, estimated from 0.009-0.01. There were also no stop codon, frameshift or non-frameshift mutations observed in the E genes of any lineage. No significant correlations between the accumulation of deleterious mutations or increasing genetic diversity and lineage extinction were observed (p>0.5). Although our hypothesis that accumulation of deleterious mutations over time led to the extinction and replacement of DENV lineages was ultimately not supported by the data, our data does highlight the significant technical issues that must be resolved in the way in which population diversity is measured for DENV and other viruses. The results provide an insight into the within-population genetic structure and diversity of DENV-1 populations.
| S-EPMC4643897 | biostudies-literature
Project description:BACKGROUND: Dengue is the world's most common mosquito-borne viral disease. Poor proofreading by RNA polymerase during its replication results in the accumulation of mutations in its genome. This leads to a diversity of genotypes in the viral population termed quasispecies. Quasispecies play an important role in disease severity. The study of quasispecies in dengue has been hindered because of the requirement for large amounts of cloning and sequencing, which could be overcome by 454 pyrosequencing. In this study, we attempted to demonstrate the feasibility of using 454 pyrosequencing to study genome diversity of dengue virus quasispecies by sequencing a pool of known dengue viral strains. RESULTS: Two sets of dengue DNA templates were sequenced by 454/Roche GS FLX. The total number of reads for data 1 and data 2 were 54,440 and 134,441, with average lengths of 221 and 232 bp, respectively. Reads containing ambiguous base Ns were excluded (6.00% in data 1, 7.05% in data 2). More than 99% of reads could be aligned back to the correct serotypes by BLAST. The reads covered the whole genome without any gaps, and the minimum coverage depth was 50×. Frequencies of known strains detected from each data set were highly correlated with the input ratios. We also explored criteria for filtering error reads and artifacts from true variations. CONCLUSIONS: This study showed that 454 pyrosequencing, coupled with our analysis procedure, could sequence the whole genome of dengue virus with good coverage. The ratio of detected variants in the sequencing data reflected the starting ratio, proving that the proposed technique could be used to study the frequencies of variants in quasispecies.
| S-EPMC3521222 | biostudies-literature
Project description:BACKGROUND: Many potentially life-threatening infectious viruses are highly mutable in nature. Characterizing the fittest variants within a quasispecies from infected patients is expected to allow unprecedented opportunities to investigate the relationship between quasispecies diversity and disease epidemiology. The advent of next-generation sequencing technologies has allowed the study of virus diversity with high-throughput sequencing, although these methods come with higher rates of errors which can artificially increase diversity. RESULTS: Here we introduce a novel computational approach that incorporates base quality scores from next-generation sequencers for reconstructing viral genome sequences that simultaneously infers the number of variants within a quasispecies that are present. Comparisons on simulated and clinical data on dengue virus suggest that the novel approach provides a more accurate inference of the underlying number of variants within the quasispecies, which is vital for clinical efforts in mapping the within-host viral diversity. Sequence alignments generated by our approach are also found to exhibit lower rates of error. CONCLUSIONS: The ability to infer the viral quasispecies colony that is present within a human host provides the potential for a more accurate classification of the viral phenotype. Understanding the genomics of viruses will be relevant not just to studying how to control or even eradicate these viral infectious diseases, but also in learning about the innate protection in the human host against the viruses.
| S-EPMC4234478 | biostudies-literature
Project description:MOTIVATION: Throughout the recent years, 454 pyrosequencing has emerged as an efficient alternative to traditional Sanger sequencing and is widely used in both de novo whole-genome sequencing and metagenomics. Especially the latter application is extremely sensitive to sequencing errors and artificially duplicated reads. Both are common in 454 pyrosequencing and can create a strong bias in the estimation of diversity and composition of a sample. To date, there are several tools that aim to remove both sequencing noise and duplicates. Nevertheless, duplicate removal is often based on nucleotide sequences rather than on the underlying flow values, which contain additional information. RESULTS: With the novel tool JATAC, we present an approach towards a more accurate duplicate removal by analysing flow values directly. Making use of previous findings on 454 flow data characteristics, we combine read clustering with Bayesian distance measures. Finally, we provide a benchmark with an existing algorithm. AVAILABILITY: JATAC is freely available under the General Public License from http://malde.org/ketil/jatac/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
| S-EPMC3605598 | biostudies-literature
Project description:BackgroundRNA viruses infecting a host usually exist as a set of closely related sequences, referred to as quasispecies. The genomic diversity of viral quasispecies is a subject of great interest, particularly for chronic infections, since it can lead to resistance to existing therapies. High-throughput sequencing is a promising approach to characterizing viral diversity, but unfortunately standard assembly software was originally designed for single genome assembly and cannot be used to simultaneously assemble and estimate the abundance of multiple closely related quasispecies sequences.ResultsIn this paper, we introduce a new Viral Spectrum Assembler (ViSpA) method for quasispecies spectrum reconstruction and compare it with the state-of-the-art ShoRAH tool on both simulated and real 454 pyrosequencing shotgun reads from HCV and HIV quasispecies. Experimental results show that ViSpA outperforms ShoRAH on simulated error-free reads, correctly assembling 10 out of 10 quasispecies and 29 sequences out of 40 quasispecies. While ShoRAH has a significant advantage over ViSpA on reads simulated with sequencing errors due to its advanced error correction algorithm, ViSpA is better at assembling the simulated reads after they have been corrected by ShoRAH. ViSpA also outperforms ShoRAH on real 454 reads. Indeed, 7 most frequent sequences reconstructed by ViSpA from a real HCV dataset are viable (do not contain internal stop codons), and the most frequent sequence was within 1% of the actual open reading frame obtained by cloning and Sanger sequencing. In contrast, only one of the sequences reconstructed by ShoRAH is viable. On a real HIV dataset, ShoRAH correctly inferred only 2 quasispecies sequences with at most 4 mismatches whereas ViSpA correctly reconstructed 5 quasispecies with at most 2 mismatches, and 2 out of 5 sequences were inferred without any mismatches. ViSpA source code is available at http://alla.cs.gsu.edu/~software/VISPA/vispa.html.ConclusionsViSpA enables accurate viral quasispecies spectrum reconstruction from 454 pyrosequencing reads. We are currently exploring extensions applicable to the analysis of high-throughput sequencing data from bacterial metagenomic samples and ecological samples of eukaryote populations.
| S-EPMC3194189 | biostudies-literature
Project description:BackgroundThe Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models.Methodology/principal findingsThe assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp). In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS), providing a resource for population and also gene function studies.ConclusionsThis paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical "stress proteins", such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of "stress" genes for studies understanding marine ectotherms' capacities to cope with environmental change.
| S-EPMC3017093 | biostudies-literature
Project description:Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785) were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx) encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%). The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases.
| S-EPMC3954088 | biostudies-literature
Project description:454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.
| S-EPMC3788733 | biostudies-literature
Project description:BackgroundThe rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments.ResultsWe obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.ConclusionsThe pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.
| S-EPMC3116506 | biostudies-literature