scholarly journals De novo long-read assembly of a complex animal genome

2017 ◽  
Author(s):  
David Eccles ◽  
Jodie Chandler ◽  
Mali Camberis ◽  
Benard Henrissat ◽  
Sergey Koren ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Assembly ◽  
Tandem Repeats ◽  
De Novo ◽  
Parasitic Nematodes ◽  
Trna Genes ◽  
Test Case ◽  
Nippostrongylus Brasiliensis ◽  
Dna Repeats ◽  
Long Read

AbstractEukaryotic genome assembly remains a challenge in part because of the prevalence of complex DNA repeats. This is a particularly acute problem for holocentric nematodes because of the large number of satellite DNA sequences found throughout their genomes. These have been recalcitrant to most genome sequencing methods. At the same time, many nematodes are parasites and some represent a serious threat to human health. There is a pressing need for better molecular characterization of animal and plant parasitic nematodes. The advent of long-read DNA sequencing methods offers the promise of resolving complex genomes. Using Nippostrongylus brasiliensis as a test case, applying improved base-calling algorithms and assembly methods, we demonstrate the feasibility of de novo genome assembly matching current community standards using only MinION long reads. In doing so, we uncovered an unexpected diversity of very long and complex DNA repeat sequences, including massive tandem repeats of tRNA genes. The method has the added advantage of preserving haplotypic variants and so has the potential to be used in population analyses.

scholarly journals Using MinION nanopore sequencing to generate a de novo eukaryotic draft genome: preliminary physiological and genomic description of the extremophilic red alga Galdieria sulphuraria strain SAG 107.79

2016 ◽  
Author(s):  
Amanda M. Davis ◽  
Manuela Iovinella ◽  
Sally James ◽  
Thomas Robshaw ◽  
Jennifer R. Dodson ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Assembly ◽  
De Novo ◽  
Sequence Data ◽  
Draft Genome ◽  
Carbon Sources ◽  
Red Alga ◽  
Full Genome Sequencing ◽  
Galdieria Sulphuraria ◽  
Long Read

AbstractWe report here the de novo assembly of a eukaryotic genome using only MinION nanopore DNA sequence data by examining a novel Galdieria sulphuraria genome: strain SAG 107.79. This extremophilic red alga was targeted for full genome sequencing as we found that it could grow on a wide variety of carbon sources and could uptake several precious and rare-earth metals, which places it as an interesting biological target for disparate industrial biotechnological uses. Phylogenetic analysis clearly places this as a species of G. sulphuraria. Here we additionally show that the genome assembly generated via nanopore long read data was of a high quality with regards to low total number of contiguous DNA sequences and long length of assemblies. Collectively, the MinION platform looks to rival other competing approaches for de novo genome acquisition with available informatics tools for assembly. The genome assembly is publically released as NCBI BioProject PRJNA330791. Further work is needed to reduce small insertion-deletion errors, relative to short-read assemblies.

scholarly journals Human Genome Assembly in 100 Minutes

2019 ◽  
Author(s):  
Chen-Shan Chin ◽  
Asif Khalak
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Genome Assembly ◽  
De Novo ◽  
Critical Factor ◽  
Read Length ◽  
De Novo Genome Assembly ◽  
Small Indels ◽  
Sequencing Technologies ◽  
Long Read

AbstractDe novo genome assembly provides comprehensive, unbiased genomic information and makes it possible to gain insight into new DNA sequences not present in reference genomes. Many de novo human genomes have been published in the last few years, leveraging a combination of inexpensive short-read and single-molecule long-read technologies. As long-read DNA sequencers become more prevalent, the computational burden of generating assemblies persists as a critical factor. The most common approach to long-read assembly, using an overlap-layout-consensus (OLC) paradigm, requires all-to-all read comparisons, which quadratically scales in computational complexity with the number of reads. We assert that recently achievements in sequencing technology (i.e. with accuracy ~99% and read length ~10-15k) enables a fundamentally better strategy for OLC that is effectively linear rather than quadratic. Our genome assembly implementation, Peregrine uses sparse hierarchical minimizers (SHIMMER) to index reads thereby avoiding the need for an all-to-all read comparison step. Peregrine can assemble 30x human PacBio CCS read datasets in less than 30 CPU hours and around 100 wall-clock minutes to a high contiguity assembly (N50 > 20Mb). The continued advance of sequencing technologies coupled with the Peregrine assembler enables routine generation of human de novo assemblies. This will allow for population scale measurements of more comprehensive genomic variations -- beyond SNPs and small indels -- as well as novel applications requiring rapid access to de novo assemblies.

Highly Condensed Potato Pericentromeric Heterochromatin Contains rDNA-Related Tandem Repeats

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1435-1444 ◽  
Author(s):  
Robert M Stupar ◽  
Junqi Song ◽  
Ahmet L Tek ◽  
Zhukuan Cheng ◽  
Fenggao Dong ◽  
...  
Keyword(s):  
Repetitive Dna ◽  
Dna Sequences ◽  
Tandem Repeats ◽  
Intergenic Spacer ◽  
Pericentromeric Heterochromatin ◽  
Dna Repeats ◽  
Solanum Bulbocastanum ◽  
Origin And Evolution ◽  
Potato Genome ◽  
Dna Elements

Abstract The heterochromatin in eukaryotic genomes represents gene-poor regions and contains highly repetitive DNA sequences. The origin and evolution of DNA sequences in the heterochromatic regions are poorly understood. Here we report a unique class of pericentromeric heterochromatin consisting of DNA sequences highly homologous to the intergenic spacer (IGS) of the 18S•25S ribosomal RNA genes in potato. A 5.9-kb tandem repeat, named 2D8, was isolated from a diploid potato species Solanum bulbocastanum. Sequence analysis indicates that the 2D8 repeat is related to the IGS of potato rDNA. This repeat is associated with highly condensed pericentromeric heterochromatin at several hemizygous loci. The 2D8 repeat is highly variable in structure and copy number throughout the Solanum genus, suggesting that it is evolutionarily dynamic. Additional IGS-related repetitive DNA elements were also identified in the potato genome. The possible mechanism of the origin and evolution of the IGS-related repeats is discussed. We demonstrate that potato serves as an interesting model for studying repetitive DNA families because it is propagated vegetatively, thus minimizing the meiotic mechanisms that can remove novel DNA repeats.

scholarly journals Chromosome-level assembly of Drosophila bifasciata reveals important karyotypic transition of the X chromosome

2019 ◽  
Author(s):  
Ryan Bracewell ◽  
Anita Tran ◽  
Kamalakar Chatla ◽  
Doris Bachtrog
Keyword(s):  
X Chromosome ◽  
Genome Assembly ◽  
De Novo ◽  
Pericentromeric Region ◽  
Species Group ◽  
Chromosome 15 ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Long Read ◽  
Chromosome Level

ABSTRACTThe Drosophila obscura species group is one of the most studied clades of Drosophila and harbors multiple distinct karyotypes. Here we present a de novo genome assembly and annotation of D. bifasciata, a species which represents an important subgroup for which no high-quality chromosome-level genome assembly currently exists. We combined long-read sequencing (Nanopore) and Hi-C scaffolding to achieve a highly contiguous genome assembly approximately 193Mb in size, with repetitive elements constituting 30.1% of the total length. Drosophila bifasciata harbors four large metacentric chromosomes and the small dot, and our assembly contains each chromosome in a single scaffold, including the highly repetitive pericentromere, which were largely composed of Jockey and Gypsy transposable elements. We annotated a total of 12,821 protein-coding genes and comparisons of synteny with D. athabasca orthologs show that the large metacentric pericentromeric regions of multiple chromosomes are conserved between these species. Importantly, Muller A (X chromosome) was found to be metacentric in D. bifasciata and the pericentromeric region appears homologous to the pericentromeric region of the fused Muller A-AD (XL and XR) of pseudoobscura/affinis subgroup species. Our finding suggests a metacentric ancestral X fused to a telocentric Muller D and created the large neo-X (Muller A-AD) chromosome ∼15 MYA. We also confirm the fusion of Muller C and D in D. bifasciata and show that it likely involved a centromere-centromere fusion.

scholarly journals Accurate long-read de novo assembly evaluation with Inspector

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yu Chen ◽  
Yixin Zhang ◽  
Amy Y. Wang ◽  
Min Gao ◽  
Zechen Chong
Keyword(s):  
Genome Assembly ◽  
De Novo Assembly ◽  
In Silico ◽  
Large Scale ◽  
De Novo ◽  
Small Scale ◽  
De Novo Genome Assembly ◽  
Consensus Sequences ◽  
Assembly Evaluation ◽  
Long Read

AbstractLong-read de novo genome assembly continues to advance rapidly. However, there is a lack of effective tools to accurately evaluate the assembly results, especially for structural errors. We present Inspector, a reference-free long-read de novo assembly evaluator which faithfully reports types of errors and their precise locations. Notably, Inspector can correct the assembly errors based on consensus sequences derived from raw reads covering erroneous regions. Based on in silico and long-read assembly results from multiple long-read data and assemblers, we demonstrate that in addition to providing generic metrics, Inspector can accurately identify both large-scale and small-scale assembly errors.

scholarly journals LRSim: a Linked Reads Simulator generating insights for better genome partitioning

2017 ◽  
Author(s):  
Ruibang Luo ◽  
Fritz J. Sedlazeck ◽  
Charlotte A. Darby ◽  
Stephen M. Kelly ◽  
Michael C. Schatz
Keyword(s):  
Genome Assembly ◽  
Fragment Length ◽  
De Novo ◽  
Library Preparation ◽  
Haplotype Phasing ◽  
Multiple Datasets ◽  
Long Read ◽  
Fine Control ◽  
Informatics Tools ◽  
Sequencing Process

AbstractMotivationLinked reads are a form of DNA sequencing commercialized by 10X Genomics that uses highly multiplexed barcoding within microdroplets to tag short reads to progenitor molecules. The linked reads, spanning tens to hundreds of kilobases, offer an alternative to long-read sequencing for de novo assembly, haplotype phasing and other applications. However, there is no available simulator, making it difficult to measure their capability or develop new informatics tools.ResultsOur analysis of 13 real linked read datasets revealed their characteristics of barcodes, molecules and partitions. Based on this, we introduce LRSim that simulates linked reads by emulating the library preparation and sequencing process with fine control of 1) the number of simulated variants; 2) the linked-read characteristics; and 3) the Illumina reads profile. We conclude from the phasing and genome assembly of multiple datasets, recommendations on coverage, fragment length, and partitioning when sequencing human and non-human genome.AvailabilityLRSIM is under MIT license and is freely available at https://github.com/aquaskyline/[email protected]

scholarly journals Fully phased human genome assembly without parental data using single-cell strand sequencing and long reads

2020 ◽  
Author(s):  
David Porubsky ◽  
◽  
Peter Ebert ◽  
Peter A. Audano ◽  
Mitchell R. Vollger ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Assembly ◽  
De Novo ◽  
Error Rates ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
De Novo Genome Assembly ◽  
Parental Data ◽  
Human Genome Assembly ◽  
Long Read

AbstractHuman genomes are typically assembled as consensus sequences that lack information on parental haplotypes. Here we describe a reference-free workflow for diploid de novo genome assembly that combines the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing1,2 with continuous long-read or high-fidelity3 sequencing data. Employing this strategy, we produced a completely phased de novo genome assembly for each haplotype of an individual of Puerto Rican descent (HG00733) in the absence of parental data. The assemblies are accurate (quality value > 40) and highly contiguous (contig N50 > 23 Mbp) with low switch error rates (0.17%), providing fully phased single-nucleotide variants, indels and structural variants. A comparison of Oxford Nanopore Technologies and Pacific Biosciences phased assemblies identified 154 regions that are preferential sites of contig breaks, irrespective of sequencing technology or phasing algorithms.

scholarly journals Chromosome-Level Assembly of Drosophila bifasciata Reveals Important Karyotypic Transition of the X Chromosome

2020 ◽  
Vol 10 (3) ◽  
pp. 891-897 ◽  
Author(s):  
Ryan Bracewell ◽  
Anita Tran ◽  
Kamalakar Chatla ◽  
Doris Bachtrog
Keyword(s):  
X Chromosome ◽  
Genome Assembly ◽  
De Novo ◽  
Pericentromeric Region ◽  
Species Group ◽  
Chromosome 15 ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Long Read ◽  
Chromosome Level

The Drosophila obscura species group is one of the most studied clades of Drosophila and harbors multiple distinct karyotypes. Here we present a de novo genome assembly and annotation of D. bifasciata, a species which represents an important subgroup for which no high-quality chromosome-level genome assembly currently exists. We combined long-read sequencing (Nanopore) and Hi-C scaffolding to achieve a highly contiguous genome assembly approximately 193 Mb in size, with repetitive elements constituting 30.1% of the total length. Drosophila bifasciata harbors four large metacentric chromosomes and the small dot, and our assembly contains each chromosome in a single scaffold, including the highly repetitive pericentromeres, which were largely composed of Jockey and Gypsy transposable elements. We annotated a total of 12,821 protein-coding genes and comparisons of synteny with D. athabasca orthologs show that the large metacentric pericentromeric regions of multiple chromosomes are conserved between these species. Importantly, Muller A (X chromosome) was found to be metacentric in D. bifasciata and the pericentromeric region appears homologous to the pericentromeric region of the fused Muller A-AD (XL and XR) of pseudoobscura/affinis subgroup species. Our finding suggests a metacentric ancestral X fused to a telocentric Muller D and created the large neo-X (Muller A-AD) chromosome ∼15 MYA. We also confirm the fusion of Muller C and D in D. bifasciata and show that it likely involved a centromere-centromere fusion.

scholarly journals Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yaoxi He ◽  
Xin Luo ◽  
Bin Zhou ◽  
Ting Hu ◽  
Xiaoyu Meng ◽  
...  
Keyword(s):  
Rhesus Macaque ◽  
Genome Assembly ◽  
De Novo ◽  
Gene Annotation ◽  
Large Body ◽  
Phenotypic Traits ◽  
Structural Variants ◽  
De Novo Genome Assembly ◽  
Chinese Rhesus Macaque ◽  
Long Read

Abstract We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.

scholarly journals A high-quality genome assembly from a single, field-collected spotted lanternfly (Lycorma delicatula) using the PacBio Sequel II system

GigaScience ◽  
2019 ◽  
Vol 8 (10) ◽  
Author(s):  
Sarah B Kingan ◽  
Julie Urban ◽  
Christine C Lambert ◽  
Primo Baybayan ◽  
Anna K Childers ◽  
...  
Keyword(s):  
Invasive Species ◽  
Genome Assembly ◽  
De Novo ◽  
Fragment Size ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Lycorma Delicatula ◽  
Long Read ◽  
Genome Assemblies ◽  
High Quality Genome

ABSTRACT Background A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies; however, long-read methods have historically had greater input DNA requirements and higher costs than next-generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female spotted lanternfly (Lycorma delicatula) using a single Pacific Biosciences SMRT Cell. The spotted lanternfly is an invasive species recently discovered in the northeastern United States that threatens to damage economically important crop plants in the region. Results The DNA from 1 individual was used to make 1 standard, size-selected library with an average DNA fragment size of ∼20 kb. The library was run on 1 Sequel II SMRT Cell 8M, generating a total of 132 Gb of long-read sequences, of which 82 Gb were from unique library molecules, representing ∼36× coverage of the genome. The assembly had high contiguity (contig N50 length = 1.5 Mb), completeness, and sequence level accuracy as estimated by conserved gene set analysis (96.8% of conserved genes both complete and without frame shift errors). Furthermore, it was possible to segregate more than half of the diploid genome into the 2 separate haplotypes. The assembly also recovered 2 microbial symbiont genomes known to be associated with L. delicatula, each microbial genome being assembled into a single contig. Conclusions We demonstrate that field-collected arthropods can be used for the rapid generation of high-quality genome assemblies, an attractive approach for projects on emerging invasive species, disease vectors, or conservation efforts of endangered species.

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