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 NanoGalaxy: Nanopore long-read sequencing data analysis in Galaxy

GigaScience ◽  
2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Willem de Koning ◽  
Milad Miladi ◽  
Saskia Hiltemann ◽  
Astrid Heikema ◽  
John P Hays ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Bioinformatics Analysis ◽  
De Novo ◽  
Sequence Data ◽  
Ease Of Use ◽  
Easy Access ◽  
Complex Data ◽  
Sequencing Data ◽  
Long Read ◽  
Sequencing Platforms

Abstract Background Long-read sequencing can be applied to generate very long contigs and even completely assembled genomes at relatively low cost and with minimal sample preparation. As a result, long-read sequencing platforms are becoming more popular. In this respect, the Oxford Nanopore Technologies–based long-read sequencing “nanopore" platform is becoming a widely used tool with a broad range of applications and end-users. However, the need to explore and manipulate the complex data generated by long-read sequencing platforms necessitates accompanying specialized bioinformatics platforms and tools to process the long-read data correctly. Importantly, such tools should additionally help democratize bioinformatics analysis by enabling easy access and ease-of-use solutions for researchers. Results The Galaxy platform provides a user-friendly interface to computational command line–based tools, handles the software dependencies, and provides refined workflows. The users do not have to possess programming experience or extended computer skills. The interface enables researchers to perform powerful bioinformatics analysis, including the assembly and analysis of short- or long-read sequence data. The newly developed “NanoGalaxy" is a Galaxy-based toolkit for analysing long-read sequencing data, which is suitable for diverse applications, including de novo genome assembly from genomic, metagenomic, and plasmid sequence reads. Conclusions A range of best-practice tools and workflows for long-read sequence genome assembly has been integrated into a NanoGalaxy platform to facilitate easy access and use of bioinformatics tools for researchers. NanoGalaxy is freely available at the European Galaxy server https://nanopore.usegalaxy.eu with supporting self-learning training material available at https://training.galaxyproject.org.

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 LongStitch: High-quality genome assembly correction and scaffolding using long reads

2021 ◽  
Author(s):  
Lauren Coombe ◽  
Janet X Li ◽  
Theodora Lo ◽  
Johnathan Wong ◽  
Vladimir Nikolic ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Draft Genome ◽  
Model Organisms ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Long Reads ◽  
Long Read ◽  
Genomic Regions ◽  
Genome Assemblies

Background Generating high-quality de novo genome assemblies is foundational to the genomics study of model and non-model organisms. In recent years, long-read sequencing has greatly benefited genome assembly and scaffolding, a process by which assembled sequences are ordered and oriented through the use of long-range information. Long reads are better able to span repetitive genomic regions compared to short reads, and thus have tremendous utility for resolving problematic regions and helping generate more complete draft assemblies. Here, we present LongStitch, a scalable pipeline that corrects and scaffolds draft genome assemblies exclusively using long reads. Results LongStitch incorporates multiple tools developed by our group and runs in up to three stages, which includes initial assembly correction (Tigmint-long), followed by two incremental scaffolding stages (ntLink and ARKS-long). Tigmint-long and ARKS-long are misassembly correction and scaffolding utilities, respectively, previously developed for linked reads, that we adapted for long reads. Here, we describe the LongStitch pipeline and introduce our new long-read scaffolder, ntLink, which utilizes lightweight minimizer mappings to join contigs. LongStitch was tested on short and long-read assemblies of three different human individuals using corresponding nanopore long-read data, and improves the contiguity of each assembly from 2.0-fold up to 304.6-fold (as measured by NGA50 length). Furthermore, LongStitch generates more contiguous and correct assemblies compared to state-of-the-art long-read scaffolder LRScaf in most tests, and consistently runs in under five hours using less than 23GB of RAM. Conclusions Due to its effectiveness and efficiency in improving draft assemblies using long reads, we expect LongStitch to benefit a wide variety of de novo genome assembly projects. The LongStitch pipeline is freely available at https://github.com/bcgsc/longstitch.

scholarly journals Extensive genomic and transcriptomic variation defines the chromosome-scale assembly of Haemonchus contortus, a model gastrointestinal worm

2020 ◽  
Author(s):  
Stephen R. Doyle ◽  
Alan Tracey ◽  
Roz Laing ◽  
Nancy Holroyd ◽  
David Bartley ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Haemonchus Contortus ◽  
Vaccine Development ◽  
De Novo ◽  
Anthelmintic Resistance ◽  
Draft Genome ◽  
Small Ruminants ◽  
High Quality ◽  
Long Read ◽  
Genome Assemblies

AbstractBackgroundHaemonchus contortus is a globally distributed and economically important gastrointestinal pathogen of small ruminants, and has become the key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Two draft genome assemblies for H. contortus were reported in 2013, however, both were highly fragmented, incomplete, and differed from one another in important respects. While the introduction of long-read sequencing has significantly increased the rate of production and contiguity of de novo genome assemblies broadly, achieving high quality genome assemblies for small, genetically diverse, outcrossing eukaryotic organisms such as H. contortus remains a significant challenge.ResultsHere, we report using PacBio long read and OpGen and 10X Genomics long-molecule methods to generate a highly contiguous 283.4 Mbp chromosome-scale genome assembly including a resolved sex chromosome. We show a remarkable pattern of almost complete conservation of chromosome content (synteny) with Caenorhabditis elegans, but almost no conservation of gene order. Long-read transcriptome sequence data has allowed us to define coordinated transcriptional regulation throughout the life cycle of the parasite, and refine our understanding of cis- and trans-splicing relative to that observed in C. elegans. Finally, we use this assembly to give a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally.ConclusionsThe H. contortus MHco3(ISE).N1 genome assembly presented here represents the most contiguous and resolved nematode assembly outside of the Caenorhabditis genus to date, together with one of the highest-quality set of predicted gene features. These data provide a high-quality comparison for understanding the evolution and genomics of Caenorhabditis and other nematodes, and extends the experimental tractability of this model parasitic nematode in understanding pathogen biology, drug discovery and vaccine development, and important adaptive traits such as drug resistance.

scholarly journals Draft Genome Assembly of the Poultry Red Mite, Dermanyssus gallinae

2018 ◽  
Vol 7 (18) ◽  
Author(s):  
Stewart T. G. Burgess ◽  
Kathryn Bartley ◽  
Francesca Nunn ◽  
Harry W. Wright ◽  
Margaret Hughes ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Gene Prediction ◽  
Draft Genome ◽  
Egg Laying ◽  
Dermanyssus Gallinae ◽  
Poultry Red Mite ◽  
Draft Genome Assembly ◽  
Long Read

The poultry red mite, Dermanyssus gallinae, is a major worldwide concern in the egg-laying industry. Here, we report the first draft genome assembly and gene prediction of Dermanyssus gallinae, based on combined PacBio and MinION long-read de novo sequencing.

scholarly journals A reference genome sequence resource for the sugar beet root rot pathogen Aphanomyces cochlioides

2021 ◽  
Author(s):  
Jacob Botkin ◽  
Ashok K Chanda ◽  
Frank N Martin ◽  
Cory D Hirsch
Keyword(s):  
Sugar Beet ◽  
Genome Assembly ◽  
Root Rot ◽  
De Novo ◽  
Sequence Data ◽  
Aphanomyces Cochlioides ◽  
Beet Root ◽  
Long Read ◽  
Illumina Sequence ◽  
Sugar Beet Root

Aphanomyces cochlioides, the causal agent of damping-off and root rot of sugar beet (Beta vulgaris L.), is a soil-dwelling oomycete responsible for yield losses in all major sugar beet growing regions. Currently, genomic resources for A. cochlioides are limited. Here we report a de novo genome assembly using a combination of long-read MinION (Oxford Nanopore Technologies) and short-read Illumina sequence data for A. cochlioides isolate 103-1, from Breckenridge, MN. The assembled genome was 76.3 Mb, with a contig N50 of 2.6 Mb. The reference assembly was annotated and was composed of 32.1% repetitive elements and 20,274 gene models. This high-quality genome assembly of A. cochlioides will be a valuable resource for understanding genetic variation, virulence factors, and comparative genomics of this important sugar beet pathogen.

scholarly journals Draft genome of a porcupinefish, Diodon Holocanthus

2019 ◽  
Author(s):  
Mengyang Xu ◽  
Xiaoshan Su ◽  
Mengqi Zhang ◽  
Ming Li ◽  
Xiaoyun Huang ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Repetitive Sequences ◽  
Draft Genome ◽  
Single Copy ◽  
Single Individual ◽  
Protein Coding ◽  
Long Read ◽  
Phylogeny And Evolution ◽  
Downstream Analysis

AbstractThe long-spine porcupinefish, Diodon holocanthus (Diodontidae, Tetraodontiformes, Actinopterygii), also known as the freckled porcupinefish, attracts great interest of ecology and economy. Its distinct characteristics including inflation reaction, spiny skin and tetradotoxin, however, have not been fully studied without a complete genome assembly.In this study, the whole genome of a single individual was sequenced using single tube-Long Fragment Read co-barcode reads, generating 154.3 Gb of paired-end data (219.8× depth). The gap was further filled using small amount of Oxford Nanopore MinION long read dataset (11.4Gb, 15.9× depth). Taking full use of long, medium, short-range of genome assembly information, the final assembled sequences with a total length of 650.02 Mb obtained contig and scaffold N50 sizes of 2.15 Mb and 8.13 Mb, respectively, despite of high repetitive content. Benchmarking Universal Single-Copy Orthologs captured 95.7% (2,474) of core genes to assess the completeness. In addition, 206.5 Mb (32.10%) of repetitive sequences were identified, and 20,840 protein-coding genes were annotated, among which 18,281 (87.72%) proteins were assigned with possible functions.This is the first demonstration of de novo genome of the porcupinefish, which will benefit downstream analysis of ontogeny, phylogeny, and evolution, and improve the exploration of its unique defensive mechanism.

scholarly journals LongStitch: high-quality genome assembly correction and scaffolding using long reads

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Lauren Coombe ◽  
Janet X. Li ◽  
Theodora Lo ◽  
Johnathan Wong ◽  
Vladimir Nikolic ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Draft Genome ◽  
Model Organisms ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Long Reads ◽  
Long Read ◽  
Genomic Regions ◽  
Genome Assemblies

Abstract Background Generating high-quality de novo genome assemblies is foundational to the genomics study of model and non-model organisms. In recent years, long-read sequencing has greatly benefited genome assembly and scaffolding, a process by which assembled sequences are ordered and oriented through the use of long-range information. Long reads are better able to span repetitive genomic regions compared to short reads, and thus have tremendous utility for resolving problematic regions and helping generate more complete draft assemblies. Here, we present LongStitch, a scalable pipeline that corrects and scaffolds draft genome assemblies exclusively using long reads. Results LongStitch incorporates multiple tools developed by our group and runs in up to three stages, which includes initial assembly correction (Tigmint-long), followed by two incremental scaffolding stages (ntLink and ARKS-long). Tigmint-long and ARKS-long are misassembly correction and scaffolding utilities, respectively, previously developed for linked reads, that we adapted for long reads. Here, we describe the LongStitch pipeline and introduce our new long-read scaffolder, ntLink, which utilizes lightweight minimizer mappings to join contigs. LongStitch was tested on short and long-read assemblies of Caenorhabditis elegans, Oryza sativa, and three different human individuals using corresponding nanopore long-read data, and improves the contiguity of each assembly from 1.2-fold up to 304.6-fold (as measured by NGA50 length). Furthermore, LongStitch generates more contiguous and correct assemblies compared to state-of-the-art long-read scaffolder LRScaf in most tests, and consistently improves upon human assemblies in under five hours using less than 23 GB of RAM. Conclusions Due to its effectiveness and efficiency in improving draft assemblies using long reads, we expect LongStitch to benefit a wide variety of de novo genome assembly projects. The LongStitch pipeline is freely available at https://github.com/bcgsc/longstitch.

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.

scholarly journals A long reads-based de-novo assembly of the genome of the Arlee homozygous line reveals chromosomal rearrangements in rainbow trout

2021 ◽  
Author(s):  
Guangtu Gao ◽  
Susana Magadan ◽  
Geoffrey C Waldbieser ◽  
Ramey C Youngblood ◽  
Paul A Wheeler ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Chromosome Number ◽  
Genome Assembly ◽  
De Novo Assembly ◽  
De Novo ◽  
Sequence Data ◽  
Structural Variations ◽  
High Coverage ◽  
Haploid Chromosome Number ◽  
Long Reads

Abstract Currently, there is still a need to improve the contiguity of the rainbow trout reference genome and to use multiple genetic backgrounds that will represent the genetic diversity of this species. The Arlee doubled haploid line was originated from a domesticated hatchery strain that was originally collected from the northern California coast. The Canu pipeline was used to generate the Arlee line genome de-novo assembly from high coverage PacBio long-reads sequence data. The assembly was further improved with Bionano optical maps and Hi-C proximity ligation sequence data to generate 32 major scaffolds corresponding to the karyotype of the Arlee line (2 N = 64). It is composed of 938 scaffolds with N50 of 39.16 Mb and a total length of 2.33 Gb, of which ∼95% was in 32 chromosome sequences with only 438 gaps between contigs and scaffolds. In rainbow trout the haploid chromosome number can vary from 29 to 32. In the Arlee karyotype the haploid chromosome number is 32 because chromosomes Omy04, 14 and 25 are divided into six acrocentric chromosomes. Additional structural variations that were identified in the Arlee genome included the major inversions on chromosomes Omy05 and Omy20 and additional 15 smaller inversions that will require further validation. This is also the first rainbow trout genome assembly that includes a scaffold with the sex-determination gene (sdY) in the chromosome Y sequence. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes on chromosomes Omy12 and Omy13.

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