SV2: accurate structural variation genotyping and de novo mutation detection from whole genomes.
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ABSTRACT:
SUBMITTER: Antaki D
PROVIDER: S-EPMC5946924 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MotivationAn accurate genome assembly from short read sequencing data is critical for downstream analysis, for example allowing investigation of variants within a sequenced population. However, assembling sequencing data from virus samples, especially RNA viruses, into a genome sequence is challenging due to the combination of viral population diversity and extremely uneven read depth caused by amplification bias in the inevitable reverse transcription and polymerase chain reaction amplification process of current methods.ResultsWe developed a new de novo assembler called IVA (Iterative Virus Assembler) designed specifically for read pairs sequenced at highly variable depth from RNA virus samples. We tested IVA on datasets from 140 sequenced samples from human immunodeficiency virus-1 or influenza-virus-infected people and demonstrated that IVA outperforms all other virus de novo assemblers.Availability and implementationThe software runs under Linux, has the GPLv3 licence and is freely available from http://sanger-pathogens.github.io/iva
| S-EPMC4495290 | biostudies-literature
Project description:BackgroundPrevious studies exploring sequence variation in the model legume, Medicago truncatula, relied on mapping short reads to a single reference. However, read-mapping approaches are inadequate to examine large, diverse gene families or to probe variation in repeat-rich or highly divergent genome regions. De novo sequencing and assembly of M. truncatula genomes enables near-comprehensive discovery of structural variants (SVs), analysis of rapidly evolving gene families, and ultimately, construction of a pan-genome.ResultsGenome-wide synteny based on 15 de novo M. truncatula assemblies effectively detected different types of SVs indicating that as much as 22% of the genome is involved in large structural changes, altogether affecting 28% of gene models. A total of 63 million base pairs (Mbp) of novel sequence was discovered, expanding the reference genome space for Medicago by 16%. Pan-genome analysis revealed that 42% (180 Mbp) of genomic sequences is missing in one or more accession, while examination of de novo annotated genes identified 67% (50,700) of all ortholog groups as dispensable - estimates comparable to recent studies in rice, maize and soybean. Rapidly evolving gene families typically associated with biotic interactions and stress response were found to be enriched in the accession-specific gene pool. The nucleotide-binding site leucine-rich repeat (NBS-LRR) family, in particular, harbors the highest level of nucleotide diversity, large effect single nucleotide change, protein diversity, and presence/absence variation. However, the leucine-rich repeat (LRR) and heat shock gene families are disproportionately affected by large effect single nucleotide changes and even higher levels of copy number variation.ConclusionsAnalysis of multiple M. truncatula genomes illustrates the value of de novo assemblies to discover and describe structural variation, something that is often under-estimated when using read-mapping approaches. Comparisons among the de novo assemblies also indicate that different large gene families differ in the architecture of their structural variation.
| S-EPMC5369179 | biostudies-literature
Project description:The discovery of genetic variation and the assembly of genome sequences are both inextricably linked to advances in DNA-sequencing technology. Short-read massively parallel sequencing has revolutionized our ability to discover genetic variation but is insufficient to generate high-quality genome assemblies or resolve most structural variation. Full resolution of variation is only guaranteed by complete de novo assembly of a genome. Here, we review approaches to genome assembly, the nature of gaps or missing sequences, and biases in the assembly process. We describe the challenges of generating a complete de novo genome assembly using current technologies and the impact that being able to perfectly sequence the genome would have on understanding human disease and evolution. Finally, we summarize recent technological advances that improve both contiguity and accuracy and emphasize the importance of complete de novo assembly as opposed to read mapping as the primary means to understanding the full range of human genetic variation.
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Project description:Building a population-specific catalogue of single nucleotide variants (SNVs), indels and structural variants (SVs) with frequencies, termed a national pan-genome, is critical for further advancing clinical and public health genetics in large cohorts. Here we report a Danish pan-genome obtained from sequencing 10 trios to high depth (50 × ). We report 536k novel SNVs and 283k novel short indels from mapping approaches and develop a population-wide de novo assembly approach to identify 132k novel indels larger than 10 nucleotides with low false discovery rates. We identify a higher proportion of indels and SVs than previous efforts showing the merits of high coverage and de novo assembly approaches. In addition, we use trio information to identify de novo mutations and use a probabilistic method to provide direct estimates of 1.27e-8 and 1.5e-9 per nucleotide per generation for SNVs and indels, respectively.
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Project description:Whole-genome sequencing has revolutionized the study of genetics. Genotyping-by-sequencing is now a viable method of genotyping, yet the bioinformatics involved can be daunting if not prohibitive for some laboratories. Here we present ArrayMaker, a user-friendly tool that extracts accurate single nucleotide polymorphism genotypes at pre-defined loci from whole-genome alignments and presents them in a standard genotyping format compatible with association analysis software and datasets genotyped on commercial array platforms. Using this tool, geneticists with only basic computing ability can genotype samples at any desired list of markers, facilitating genome-wide association analysis, fine mapping, candidate variant assessment, data sharing and compatibility of data sourced from multiple technologies.ArrayMaker is licensed under The MIT License and can be freely obtained at https://github.com/cw2014/ArrayMaker/. The program is implemented in Perl and runs on Linux operating systems.Supplementary data are available at Bioinformatics online.cali.willet@sydney.edu.au.
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Project description:In the last decade, one of the biggest challenges in genomics research has been to distinguish definitive pathogenic variants from all likely pathogenic variants identified by next-generation sequencing. This task is particularly complex because of our lack of knowledge regarding overall genome variation and pathogenicity of the variants. Therefore, obtaining sufficient information about genome variants in the general population is necessary as such data could be used for the interpretation of de novo mutations (DNMs) in the context of patient's phenotype in cases of sporadic genetic disease. In this study, data from whole-exome sequencing of the general population in Lithuania were directly examined. In total, 84 (VarScan) and 95 (VarSeqTM) DNMs were identified and validated using different algorithms. Thirty-nine of these mutations were considered likely to be pathogenic based on gene function, evolutionary conservation, and mutation impact. The mutation rate estimated per position pair per generation was 2.74 × 10-8 [95% CI: 2.24 × 10-8-3.35 × 10-8] (VarScan) and 2.4 × 10-8 [95% CI: 1.96 × 10-8-2.99 × 10-8] (VarSeqTM), with 1.77 × 10-8 [95% CI: 6.03 × 10-9-5.2 × 10-8] de novo indels per position per generation. The rate of germline DNMs in the Lithuanian population and the effects of the genomic and epigenetic context on DNM formation were calculated for the first time in this study, providing a basis for further analysis of DNMs in individuals with genetic diseases. Considering these findings, additional studies in patient groups with genetic diseases with unclear etiology may facilitate our ability to distinguish certain pathogenic or adaptive DNMs from tolerated background DNMs and to reliably identify disease-causing DNMs by their properties through direct observation.
| S-EPMC6102505 | biostudies-literature
Project description:The evolution in next-generation sequencing (NGS) technology has led to the development of many different assembly algorithms, but few of them focus on assembling the organelle genomes. These genomes are used in phylogenetic studies, food identification and are the most deposited eukaryotic genomes in GenBank. Producing organelle genome assembly from whole genome sequencing (WGS) data would be the most accurate and least laborious approach, but a tool specifically designed for this task is lacking. We developed a seed-and-extend algorithm that assembles organelle genomes from whole genome sequencing (WGS) data, starting from a related or distant single seed sequence. The algorithm has been tested on several new (Gonioctena intermedia and Avicennia marina) and public (Arabidopsis thaliana and Oryza sativa) whole genome Illumina data sets where it outperforms known assemblers in assembly accuracy and coverage. In our benchmark, NOVOPlasty assembled all tested circular genomes in less than 30 min with a maximum memory requirement of 16 GB and an accuracy over 99.99%. In conclusion, NOVOPlasty is the sole de novo assembler that provides a fast and straightforward extraction of the extranuclear genomes from WGS data in one circular high quality contig. The software is open source and can be downloaded at https://github.com/ndierckx/NOVOPlasty.
| S-EPMC5389512 | biostudies-literature
Project description:BACKGROUND: De novo genome assembly can be challenging due to inherent properties of the reads, even when using current state-of-the-art assembly tools based on de Bruijn graphs. Often users are not bio-informaticians and, in a black box approach, utilise assembly parameters such as contig length and N50 to generate whole genome sequences, potentially resulting in mis-assemblies. FINDINGS: Utilising several assembly tools based on de Bruijn graphs like Velvet, SPAdes and IDBA, we demonstrate that at the optimal N50, mis-assemblies do occur, even when using the multi-k-mer approaches of SPAdes and IDBA. We demonstrate that whole genome mapping can be used to identify these mis-assemblies and can guide the selection of the best k-mer size which yields the highest N50 without mis-assemblies. CONCLUSIONS: We demonstrate the utility of whole genome mapping (WGM) as a tool to identify mis-assemblies and to guide k-mer selection and higher quality de novo genome assembly of bacterial genomes.
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Project description:De novo mutations are central for evolution, since they provide the raw material for natural selection by regenerating genetic variation. However, studying de novo mutations is challenging, and is generally restricted to model species, so we have a limited understanding of the evolution of the mutation rate and spectrum between closely related species. Here, we present a mutation accumulation (MA) experiment to study de novo mutation in the unicellular green alga Chlamydomonas incerta, and perform comparative analyses with its closest known relative, C. reinhardtii. Using whole-genome sequencing data, we estimate that the median single nucleotide mutation (SNM) rate in C. incerta is μ = 7.6 x 10-10, and is highly variable between MA lines, ranging from μ = 0.35 x 10-10 to μ = 131.7 x 10-10. The SNM rate is strongly positively correlated with the mutation rate for insertions and deletions between lines (r > 0.97). We infer that the genomic factors associated with variation in the mutation rate are similar to those in C. reinhardtii, allowing for cross-prediction between species. Among these genomic factors, sequence context and complexity are more important than GC content. With the exception of a remarkably high C→T bias, the SNM spectrum differs markedly between the two Chlamydomonas species. Our results suggest that similar genomic and biological characteristics may result in a similar mutation rate in the two species, whereas the SNM spectrum has more freedom to diverge.
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Project description:Genome assemblies that are accurate, complete and contiguous are essential for identifying important structural and functional elements of genomes and for identifying genetic variation. Nevertheless, most recent genome assemblies remain incomplete and fragmented. While long molecule sequencing promises to deliver more complete genome assemblies with fewer gaps, concerns about error rates, low yields, stringent DNA requirements and uncertainty about best practices may discourage many investigators from adopting this technology. Here, in conjunction with the platinum standard Drosophila melanogaster reference genome, we analyze recently published long molecule sequencing data to identify what governs completeness and contiguity of genome assemblies. We also present a hybrid meta-assembly approach that achieves remarkable assembly contiguity for both Drosophila and human assemblies with only modest long molecule sequencing coverage. Our results motivate a set of preliminary best practices for obtaining accurate and contiguous assemblies, a 'missing manual' that guides key decisions in building high quality de novo genome assemblies, from DNA isolation to polishing the assembly.
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