scholarly journals De novo reconstruction of microbial haplotypes by integrating statistical and physical linkage

2020 ◽  
Author(s):  
Chen Cao ◽  
Jingni He ◽  
Lauren Mak ◽  
Deshan Perera ◽  
Devin Kwok ◽  
...  
Keyword(s):  
Single Molecule ◽  
Human Genetics ◽  
De Novo ◽  
Real Data ◽  
Sequencing Technologies ◽  
Physical Linkage ◽  
Pooled Sequencing ◽  
Computational Reconstruction ◽  
Host Genetic ◽  
Host Evolution

ABSTRACTDNA sequencing technologies provide unprecedented opportunities to analyze within-host evolution of microorganism populations. Often, within-host populations are analyzed via pooled sequencing of the population, which contains multiple individuals or ‘haplotypes’. However, current next-generation sequencing instruments, in conjunction with single-molecule barcoded linked-reads, cannot distinguish long haplotypes directly. Computational reconstruction of haplotypes from pooled sequencing has been attempted in virology, bacterial genomics, metagenomics and human genetics, using algorithms based on either cross-host genetic sharing or within-host genomic reads. Here we describe PoolHapX, a flexible computational approach that integrates information from both genetic sharing and genomic sequencing. We demonstrated that PoolHapX outperforms state-of-the-art tools tailored to specific organismal systems, and is robust to within-host evolution. Importantly, together with barcoded linked-reads, PoolHapX can infer whole-chromosome-scale haplotypes from 50 pools each containing 12 different haplotypes. By analyzing real data, we uncovered dynamic variations in the evolutionary processes of within-patient HIV populations previously unobserved in single position-based analysis.

scholarly journals Reconstruction of Microbial Haplotypes by Integration of Statistical and Physical Linkage in Scaffolding

2021 ◽  
Author(s):  
Chen Cao ◽  
Jingni He ◽  
Lauren Mak ◽  
Deshan Perera ◽  
Devin Kwok ◽  
...  
Keyword(s):  
Single Molecule ◽  
Human Genetics ◽  
Real Data ◽  
Sequencing Technologies ◽  
Bacterial Genomics ◽  
Physical Linkage ◽  
Pooled Sequencing ◽  
Computational Reconstruction ◽  
Host Genetic ◽  
Host Evolution

Abstract DNA sequencing technologies provide unprecedented opportunities to analyze within-host evolution of microorganism populations. Often, within-host populations are analyzed via pooled sequencing of the population, which contains multiple individuals or “haplotypes.” However, current next-generation sequencing instruments, in conjunction with single-molecule barcoded linked-reads, cannot distinguish long haplotypes directly. Computational reconstruction of haplotypes from pooled sequencing has been attempted in virology, bacterial genomics, metagenomics, and human genetics, using algorithms based on either cross-host genetic sharing or within-host genomic reads. Here, we describe PoolHapX, a flexible computational approach that integrates information from both genetic sharing and genomic sequencing. We demonstrated that PoolHapX outperforms state-of-the-art tools tailored to specific organismal systems, and is robust to within-host evolution. Importantly, together with barcoded linked-reads, PoolHapX can infer whole-chromosome-scale haplotypes from 50 pools each containing 12 different haplotypes. By analyzing real data, we uncovered dynamic variations in the evolutionary processes of within-patient HIV populations previously unobserved in single position-based analysis.

scholarly journals RECORD: Reference-Assisted Genome Assembly for Closely Related Genomes

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Krisztian Buza ◽  
Bartek Wilczynski ◽  
Norbert Dojer
Keyword(s):  
Reference Genome ◽  
De Novo ◽  
Real Data ◽  
Reference Sequence ◽  
Individual Genome ◽  
Single Experiment ◽  
Sequencing Technologies ◽  
Sequencing Cost ◽  
The Individual ◽  
Assembly Software

Background. Next-generation sequencing technologies are now producing multiple times the genome size in total reads from a single experiment. This is enough information to reconstruct at least some of the differences between the individual genome studied in the experiment and the reference genome of the species. However, in most typical protocols, this information is disregarded and the reference genome is used.Results. We provide a new approach that allows researchers to reconstruct genomes very closely related to the reference genome (e.g., mutants of the same species) directly from the reads used in the experiment. Our approach applies de novo assembly software to experimental reads and so-called pseudoreads and uses the resulting contigs to generate a modified reference sequence. In this way, it can very quickly, and at no additional sequencing cost, generate new, modified reference sequence that is closer to the actual sequenced genome and has a full coverage. In this paper, we describe our approach and test its implementation called RECORD. We evaluate RECORD on both simulated and real data. We made our software publicly available on sourceforge.Conclusion. Our tests show that on closely related sequences RECORD outperforms more general assisted-assembly software.

scholarly journals CAMISIM: Simulating metagenomes and microbial communities

2018 ◽  
Author(s):  
Adrian Fritz ◽  
Peter Hofmann ◽  
Stephan Majda ◽  
Eik Dahms ◽  
Johannes Dröge ◽  
...  
Keyword(s):  
Microbial Communities ◽  
De Novo ◽  
Real Data ◽  
Small Data ◽  
Data Sets ◽  
Sequencing Data ◽  
Taxonomic Profiling ◽  
Benchmark Data ◽  
Sequencing Technologies ◽  
Wide Range

Shotgun metagenome data sets of microbial communities are highly diverse, not only due to the natural variation of the underlying biological systems, but also due to differences in laboratory protocols, replicate numbers, and sequencing technologies. Accordingly, to effectively assess the performance of metagenomic analysis software, a wide range of benchmark data sets are required. Here, we describe the CAMISIM microbial community and metagenome simulator. The software can model different microbial abundance profiles, multi-sample time series and differential abundance studies, includes real and simulated strain-level diversity, and generates second and third generation sequencing data from taxonomic profiles or de novo. Gold standards are created for sequence assembly, genome binning, taxonomic binning, and taxonomic profiling. CAMSIM generated the benchmark data sets of the first CAMI challenge. For two simulated multi-sample data sets of the human and mouse gut microbiomes we observed high functional congruence to the real data. As further applications, we investigated the effect of varying evolutionary genome divergence, sequencing depth, and read error profiles on two popular metagenome assemblers, MEGAHIT and metaSPAdes, on several thousand small data sets generated with CAMISIM. CAMISIM can simulate a wide variety of microbial communities and metagenome data sets together with truth standards for method evaluation. All data sets and the software are freely available at: https://github.com/CAMI-challenge/CAMISIM

scholarly journals Mapping and phasing of structural variation in patient genomes using nanopore sequencing

2017 ◽  
Author(s):  
Mircea Cretu Stancu ◽  
Markus J. van Roosmalen ◽  
Ivo Renkens ◽  
Marleen Nieboer ◽  
Sjors Middelkamp ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Human Genetic Disease ◽  
Structural Genomic ◽  
Short Read ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Long Read ◽  
Complex Structural

AbstractStructural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications < 200bp in size. Long sequencing reads enabled efficient phasing of genetic variations, allowing the construction of genome-wide maps of phased SVs and SNVs. We employed read-based phasing to show that all de novo chromothripsis breakpoints occurred on paternal chromosomes and we resolved the long-range structure of the chromothripsis. This work demonstrates the value of long-read sequencing for screening whole genomes of patients for complex structural variants.

scholarly journals De novo Assembly of the Brugia malayi Genome Using Long Reads from a Single MinION Flowcell

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joseph R. Fauver ◽  
John Martin ◽  
Gary J. Weil ◽  
Makedonka Mitreva ◽  
Peter U. Fischer
Keyword(s):  
Single Molecule ◽  
New Technologies ◽  
Reference Genome ◽  
De Novo ◽  
Complete Mitochondrial Genome ◽  
Nuclear Genome ◽  
Brugia Malayi ◽  
Field Isolates ◽  
Sequencing Technologies ◽  
Long Reads

AbstractFilarial nematode infections cause a substantial global disease burden. Genomic studies of filarial worms can improve our understanding of their biology and epidemiology. However, genomic information from field isolates is limited and available reference genomes are often discontinuous. Single molecule sequencing technologies can reduce the cost of genome sequencing and long reads produced from these devices can improve the contiguity and completeness of genome assemblies. In addition, these new technologies can make generation and analysis of large numbers of field isolates feasible. In this study, we assessed the performance of the Oxford Nanopore Technologies MinION for sequencing and assembling the genome of Brugia malayi, a human parasite widely used in filariasis research. Using data from a single MinION flowcell, a 90.3 Mb nuclear genome was assembled into 202 contigs with an N50 of 2.4 Mb. This assembly covered 96.9% of the well-defined B. malayi reference genome with 99.2% identity. The complete mitochondrial genome was obtained with individual reads and the nearly complete genome of the endosymbiotic bacteria Wolbachia was assembled alongside the nuclear genome. Long-read data from the MinION produced an assembly that approached the quality of a well-established reference genome using comparably fewer resources.

scholarly journals Customized de novo mutation detection for any variant calling pipeline: SynthDNM

2021 ◽  
Author(s):  
Aojie Lian ◽  
James Guevara ◽  
Kun Xia ◽  
Jonathan Sebat
Keyword(s):  
Mutation Detection ◽  
De Novo ◽  
Variant Calling ◽  
Real Data ◽  
Supplementary Information ◽  
De Novo Mutation ◽  
Flexible Approach ◽  
Sequencing Technologies ◽  
Training Examples ◽  
Simulated Training

Abstract Motivation As sequencing technologies and analysis pipelines evolve, de novo mutation (DNM) calling tools must be adapted. Therefore, a flexible approach is needed that can accurately identify DNMs from genome or exome sequences from a variety of datasets and variant calling pipelines. Results Here, we describe SynthDNM, a random-forest based classifier that can be readily adapted to new sequencing or variant-calling pipelines by applying a flexible approach to constructing simulated training examples from real data. The optimized SynthDNM classifiers predict de novo SNPs and indels with robust accuracy across multiple methods of variant calling. Availabilityand implementation SynthDNM is freely available on Github (https://github.com/james-guevara/synthdnm). Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals High-Quality Assembly of an Individual of Yoruban Descent

2016 ◽  
Author(s):  
Karyn Meltz Steinberg ◽  
Tina Graves Lindsay ◽  
Valerie A. Schneider ◽  
Mark J.P. Chaisson ◽  
Chad Tomlinson ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Bac Library ◽  
Segmental Duplications ◽  
High Quality ◽  
Sequencing Technologies ◽  
Human Genomes ◽  
Genome Assemblies ◽  
Complete Genomic

ABSTRACTDe novo assembly of human genomes is now a tractable effort due in part to advances in sequencing and mapping technologies. We use PacBio single-molecule, real-time (SMRT) sequencing and BioNano genomic maps to construct the first de novo assembly of NA19240, a Yoruban individual from Africa. This chromosome-scaffolded assembly of 3.08 Gb with a contig N50 of 7.25 Mb and a scaffold N50 of 78.6 Mb represents one of the most contiguous high-quality human genomes. We utilize a BAC library derived from NA19240 DNA and novel haplotype-resolving sequencing technologies and algorithms to characterize regions of complex genomic architecture that are normally lost due to compression to a linear haploid assembly. Our results demonstrate that multiple technologies are still necessary for complete genomic representation, particularly in regions of highly identical segmental duplications. Additionally, we show that diploid assembly has utility in improving the quality of de novo human genome assemblies.

scholarly journals Highly-accurate long-read sequencing improves variant detection and assembly of a human genome

2019 ◽  
Author(s):  
Aaron M. Wenger ◽  
Paul Peluso ◽  
William J. Rowell ◽  
Pi-Chuan Chang ◽  
Richard J. Hall ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Short Reads ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read ◽  
Variant Detection ◽  
High Quality Genome ◽  
Circular Consensus Sequencing

AbstractThe major DNA sequencing technologies in use today produce either highly-accurate short reads or noisy long reads. We developed a protocol based on single-molecule, circular consensus sequencing (CCS) to generate highly-accurate (99.8%) long reads averaging 13.5 kb and applied it to sequence the well-characterized human HG002/NA24385. We optimized existing tools to comprehensively detect variants, achieving precision and recall above 99.91% for SNVs, 95.98% for indels, and 95.99% for structural variants. We estimate that 2,434 discordances are correctable mistakes in the high-quality Genome in a Bottle benchmark. Nearly all (99.64%) variants are phased into haplotypes, which further improves variant detection. De novo assembly produces a highly contiguous and accurate genome with contig N50 above 15 Mb and concordance of 99.998%. CCS reads match short reads for small variant detection, while enabling structural variant detection and de novo assembly at similar contiguity and markedly higher concordance than noisy long reads.

scholarly journals Customized de novo mutation detection for any variant calling pipeline: SynthDNM

2021 ◽  
Author(s):  
Aojie Lian ◽  
James Guevara ◽  
Kun Xia ◽  
Jonathan Sebat
Keyword(s):  
De Novo ◽  
Variant Calling ◽  
Real Data ◽  
Supplementary Information ◽  
De Novo Mutation ◽  
De Novo Mutations ◽  
Flexible Approach ◽  
Sequencing Technologies ◽  
Training Examples ◽  
Simulated Training

AbstractMotivationAs sequencing technologies and analysis pipelines evolve, DNM calling tools must be adapted. Therefore, a flexible approach is needed that can accurately identify de novo mutations from genome or exome sequences from a variety of datasets and variant calling pipelines.ResultsHere, we describe SynthDNM, a random-forest based classifier that can be readily adapted to new sequencing or variant-calling pipelines by applying a flexible approach to constructing simulated training examples from real data. The optimized SynthDNM classifiers predict de novo SNPs and indels with robust accuracy across multiple methods of variant calling.AvailabilitySynthDNM is freely available on Github (https://github.com/james-guevara/synthdnm)[email protected] informationSupplementary data are available at Bioinformatics online.

scholarly journals de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer

2016 ◽  
Author(s):  
Benjamin Istace ◽  
Anne Friedrich ◽  
Léo d’Agata ◽  
Sébastien Faye ◽  
Emilie Payen ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Low Cost ◽  
Ease Of Use ◽  
Yeast Genome ◽  
Structural Variations ◽  
Sequencing Technologies ◽  
Population Genomic ◽  
Oxford Nanopore ◽  
Sequencing Strategy

AbstractOxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small and low-cost single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments. The Oxford Nanopore technology is truly disruptive and can sequence small genomes in a matter of seconds. It has the potential to revolutionize genomic applications due to its portability, low-cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65% of the chromosomes. This high continuity allowed us to accurately detect large structural variations across the 21 studied genomes. Moreover, because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy.

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