Dataset Information

Rapid genome assembly and comparison decode intrastrain variation in human alphaherpesviruses.

ABSTRACT: UNLABELLED:Herpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. Here we developed and applied a streamlined new approach for assembly and comparison of large DNA viral genomes such as HSV-1. This viral genome assembly (VirGA) workflow incorporates a combination of de novo assembly, alignment, and annotation strategies to automate the generation of draft genomes for large viruses. We applied this approach to quantify the amount of variation between clonal derivatives of a common parental virus stock. In addition, we examined the genetic basis for syncytial plaque phenotypes displayed by a subset of these strains. In each of the syncytial strains, we found an identical DNA change, affecting one residue in the gB (UL27) fusion protein. Since these identical mutations could have appeared after extensive in vitro passaging, we applied the VirGA sequencing and comparison approach to two clinical HSV-1 strains isolated from the same patient. One of these strains was syncytial upon first culturing; its sequence revealed the same gB mutation. These data provide insight into the extent and origin of genome-wide intrastrain HSV-1 variation and present useful methods for expansion to in vivo patient infection studies. IMPORTANCE:Herpes simplex virus (HSV) infects more than 70% of adults worldwide, causing epithelial lesions and recurrent disease that manifests over a lifetime. Prior work has demonstrated that HSV strains vary from country to country and between individuals. However, the amount of variation within one strain has not been well studied. To address this, we developed a new approach for viral genome assembly (VirGA) and analysis. We used this approach to quantify the amount of variation between sister clones of a common parental virus stock and to determine the basis of a unique fusion phenotype displayed by several variants. These data revealed that while sister clones of one HSV stock are more than 98% identical, these variants harbor enough genetic differences to change their observed characteristics. Comparative genomics approaches will allow us to explore the impacts of viral inter- and intrastrain diversity on drug and vaccine efficacy.

SUBMITTER: Parsons LR

PROVIDER: S-EPMC4453532 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Rapid genome assembly and comparison decode intrastrain variation in human alphaherpesviruses.

Parsons Lance R LR Tafuri Yolanda R YR Shreve Jacob T JT Bowen Christopher D CD Shipley Mackenzie M MM Enquist L W LW Szpara Moriah L ML

mBio 20150331 2

<h4>Unlabelled</h4>Herpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. H ...[more]

PMID: 25827418

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Project description:BackgroundMitochondria are the cell organelles that produce most of the chemical energy required to power the cell's biochemical reactions. Despite being a part of a eukaryotic host cell, the mitochondria contain a separate genome whose origin is linked with the endosymbiosis of a prokaryotic cell by the host cell and encode independent genomic information throughout their genomes. Mitochondrial genomes accommodate essential genes and are regularly utilized in biotechnology and phylogenetics. Various assemblers capable of generating complete mitochondrial genomes are being continuously developed. These tools often use whole-genome sequencing data as an input containing reads from the mitochondrial genome. Till now, no published work has explored the systematic comparison of all the available tools for assembling human mitochondrial genomes using short-read sequencing data. This evaluation is required to identify the best tool that can be well-optimized for small-scale projects or even national-level research.ResultsIn this study, we have tested the mitochondrial genome assemblers for both simulated datasets and whole genome sequencing (WGS) datasets of humans. For the highest computational setting of 16 computational threads with the simulated dataset having 1000X read depth, MitoFlex took the least execution time of 69 s, and IOGA took the longest execution time of 1278 s. NOVOPlasty utilized the least computational memory of approximately 0.098 GB for the same setting, whereas IOGA utilized the highest computational memory of 11.858 GB. In the case of WGS datasets for humans, GetOrganelle and MitoFlex performed the best in capturing the SNPs information with a mean F1-score of 0.919 at the sequencing depth of 10X. MToolBox and NOVOPlasty performed consistently across all sequencing depths with a mean F1 score of 0.897 and 0.890, respectively.ConclusionsBased on the overall performance metrics and consistency in assembly quality for all sequencing data, MToolBox performed the best. However, NOVOPlasty was the second fastest tool in execution time despite being single-threaded, and it utilized the least computational resources among all the assemblers when tested on simulated datasets. Therefore, NOVOPlasty may be more practical when there is a significant sample size and a lack of computational resources. Besides, as long-read sequencing gains popularity, mitochondrial genome assemblers must be developed to use long-read sequencing data.

Project description:Within-host evolution produces genetic diversity in bacterial strains that cause chronic human infections. However, the lack of facile methods to measure bacterial allelic variation in clinical samples has limited understanding of intrastrain diversity's effects on disease. Here, we report a new method termed genome capture sequencing (GenCap-Seq) in which users inexpensively make hybridization probes from genomic DNA or PCR amplicons to selectively enrich and sequence targeted bacterial DNA from clinical samples containing abundant human or nontarget bacterial DNA. GenCap-Seq enables accurate measurement of allele frequencies over targeted regions and is scalable from specific genes to entire genomes, including the strain-specific accessory genome. The method is effective with samples in which target DNA is rare and inhibitory and DNA-degrading substances are abundant, including human sputum and feces. In proof-of-principle experiments, we used GenCap-Seq to investigate the responses of diversified Pseudomonas aeruginosa populations chronically infecting the lungs of people with cystic fibrosis to in vivo antibiotic exposure, and we found that treatment consistently reduced intrastrain genomic diversity. In addition, analysis of gene-level allele frequency changes suggested that some genes without conventional resistance functions may be important for bacterial fitness during in vivo antibiotic exposure. GenCap-Seq's ability to scalably enrich targeted bacterial DNA from complex samples will enable studies on the effects of intrastrain and intraspecies diversity in human infectious disease. IMPORTANCE Genetic diversity evolves in bacterial strains during human infections and could affect disease manifestations and treatment resistance. However, the extent of diversity present in vivo and its changes over time are difficult to measure by conventional methods. We developed a novel approach, GenCap-Seq, to enrich microbial DNA from complex human samples like sputum and feces for genome-wide measurements of bacterial allelic diversity. The approach is inexpensive, scalable to encompass entire targeted genomes, and works in the presence of abundant untargeted nucleic acids and inhibiting substances. We used GenCap-Seq to investigate in vivo responses of diversified bacterial strains to antibiotic treatment. This method will enable new ideas about the effects of intrastrain diversity on human infections to be tested.

Project description:BACKGROUND:Structural variants (SVs) are less common than single nucleotide polymorphisms and indels in the population, but collectively account for a significant fraction of genetic polymorphism and diseases. Base pair differences arising from SVs are on a much higher order (>100 fold) than point mutations; however, none of the current detection methods are comprehensive, and currently available methodologies are incapable of providing sufficient resolution and unambiguous information across complex regions in the human genome. To address these challenges, we applied a high-throughput, cost-effective genome mapping technology to comprehensively discover genome-wide SVs and characterize complex regions of the YH genome using long single molecules (>150 kb) in a global fashion. RESULTS:Utilizing nanochannel-based genome mapping technology, we obtained 708 insertions/deletions and 17 inversions larger than 1 kb. Excluding the 59 SVs (54 insertions/deletions, 5 inversions) that overlap with N-base gaps in the reference assembly hg19, 666 non-gap SVs remained, and 396 of them (60%) were verified by paired-end data from whole-genome sequencing-based re-sequencing or de novo assembly sequence from fosmid data. Of the remaining 270 SVs, 260 are insertions and 213 overlap known SVs in the Database of Genomic Variants. Overall, 609 out of 666 (90%) variants were supported by experimental orthogonal methods or historical evidence in public databases. At the same time, genome mapping also provides valuable information for complex regions with haplotypes in a straightforward fashion. In addition, with long single-molecule labeling patterns, exogenous viral sequences were mapped on a whole-genome scale, and sample heterogeneity was analyzed at a new level. CONCLUSION:Our study highlights genome mapping technology as a comprehensive and cost-effective method for detecting structural variation and studying complex regions in the human genome, as well as deciphering viral integration into the host genome.

Dataset Information

Rapid genome assembly and comparison decode intrastrain variation in human alphaherpesviruses.

Publications

Rapid genome assembly and comparison decode intrastrain variation in human alphaherpesviruses.

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