scholarly journals Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Caroline Belser ◽  
Franc-Christophe Baurens ◽  
Benjamin Noel ◽  
Guillaume Martin ◽  
Corinne Cruaud ◽  
...  
Keyword(s):  
Musa Acuminata ◽  
Genetic Maps ◽  
Nanopore Sequencing ◽  
Genome Coverage ◽  
Long Reads ◽  
Oxford Nanopore ◽  
A Genome ◽  
Long Read ◽  
Genome Assemblies ◽  
First Time

AbstractLong-read technologies hold the promise to obtain more complete genome assemblies and to make them easier. Coupled with long-range technologies, they can reveal the architecture of complex regions, like centromeres or rDNA clusters. These technologies also make it possible to know the complete organization of chromosomes, which remained complicated before even when using genetic maps. However, generating a gapless and telomere-to-telomere assembly is still not trivial, and requires a combination of several technologies and the choice of suitable software. Here, we report a chromosome-scale assembly of a banana genome (Musa acuminata) generated using Oxford Nanopore long-reads. We generated a genome coverage of 177X from a single PromethION flowcell with near 17X with reads longer than 75 kbp. From the 11 chromosomes, 5 were entirely reconstructed in a single contig from telomere to telomere, revealing for the first time the content of complex regions like centromeres or clusters of paralogous genes.

scholarly journals Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing

2021 ◽  
Author(s):  
Caroline Belser ◽  
Franc-Christophe Baurens ◽  
Benjamin Noel ◽  
Guillaume Martin ◽  
Corinne Cruaud ◽  
...  
Keyword(s):  
Musa Acuminata ◽  
Genetic Maps ◽  
Nanopore Sequencing ◽  
Genome Coverage ◽  
Long Reads ◽  
Oxford Nanopore ◽  
A Genome ◽  
Long Read ◽  
Genome Assemblies ◽  
First Time

AbstractLong-read technologies hold the promise to obtain more complete genome assemblies and to make them easier. Coupled with long-range technologies, they can reveal the architecture of complex regions, like centromeres or rDNA clusters. These technologies also make it possible to know the complete organization of chromosomes, which remained complicated before even when using genetic maps. However, generating a gapless and telomere-to-telomere assembly is still not trivial, and requires a combination of several technologies and the choice of suitable software. Here, we report a chromosome-scale assembly of a banana genome (Musa acuminata) generated using Oxford Nanopore long-reads. We generated a genome coverage of 177X from a single PromethION flowcell with near 17X with reads longer than 75Kb. From the 11 chromosomes, 5 were entirely reconstructed in a single contig from telomere to telomere, revealing for the first time the content of complex regions like centromeres or clusters of paralogous genes.

scholarly journals Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
High Quality ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

Abstract Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

scholarly journals Evaluation of Germline Structural Variant Calling Methods for Nanopore Sequencing Data

2021 ◽  
Vol 12 ◽  
Author(s):  
Davide Bolognini ◽  
Alberto Magi
Keyword(s):  
Variant Calling ◽  
Research Report ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Factors Affecting ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Sequencing Studies ◽  
Long Read

Structural variants (SVs) are genomic rearrangements that involve at least 50 nucleotides and are known to have a serious impact on human health. While prior short-read sequencing technologies have often proved inadequate for a comprehensive assessment of structural variation, more recent long reads from Oxford Nanopore Technologies have already been proven invaluable for the discovery of large SVs and hold the potential to facilitate the resolution of the full SV spectrum. With many long-read sequencing studies to follow, it is crucial to assess factors affecting current SV calling pipelines for nanopore sequencing data. In this brief research report, we evaluate and compare the performances of five long-read SV callers across four long-read aligners using both real and synthetic nanopore datasets. In particular, we focus on the effects of read alignment, sequencing coverage, and variant allele depth on the detection and genotyping of SVs of different types and size ranges and provide insights into precision and recall of SV callsets generated by integrating the various long-read aligners and SV callers. The computational pipeline we propose is publicly available at https://github.com/davidebolo1993/EViNCe and can be adjusted to further evaluate future nanopore sequencing datasets.

scholarly journals LINKS: Scaffolding genome assemblies with kilobase-long nanopore reads

2015 ◽  
Author(s):  
Rene L Warren ◽  
Benjamin P Vandervalk ◽  
Steven JM Jones ◽  
Inanc Birol
Keyword(s):  
Genome Assembly ◽  
Error Rates ◽  
Great Promise ◽  
Genomic Information ◽  
E Coli ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
K 12 ◽  
Genome Assemblies

Owing to the complexity of the assembly problem, we do not yet have complete genome sequences. The difficulty in assembling reads into finished genomes is exacerbated by sequence repeats and the inability of short reads to capture sufficient genomic information to resolve those problematic regions. Established and emerging long read technologies show great promise in this regard, but their current associated higher error rates typically require computational base correction and/or additional bioinformatics pre-processing before they could be of value. We present LINKS, the Long Interval Nucleotide K-mer Scaffolder algorithm, a solution that makes use of the information in error-rich long reads, without the need for read alignment or base correction. We show how the contiguity of an ABySS E. coli K-12 genome assembly could be increased over five-fold by the use of beta-released Oxford Nanopore Ltd. (ONT) long reads and how LINKS leverages long-range information in S. cerevisiae W303 ONT reads to yield an assembly with less than half the errors of competing applications. Re-scaffolding the colossal white spruce assembly draft (PG29, 20 Gbp) and how LINKS scales to larger genomes is also presented. We expect LINKS to have broad utility in harnessing the potential of long reads in connecting high-quality sequences of small and large genome assembly drafts. Availability: http://www.bcgsc.ca/bioinfo/software/links

scholarly journals Benchmarking Long-Read Assemblers for Genomic Analyses of Bacterial Pathogens Using Oxford Nanopore Sequencing

2020 ◽  
Vol 21 (23) ◽  
pp. 9161
Author(s):  
Zhao Chen ◽  
David L. Erickson ◽  
Jianghong Meng
Keyword(s):  
Virulence Genes ◽  
Bacterial Pathogens ◽  
Error Rates ◽  
Nanopore Sequencing ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Genomic Analyses ◽  
Long Read ◽  
Genome Analyses ◽  
Assembly Algorithms

Oxford Nanopore sequencing can be used to achieve complete bacterial genomes. However, the error rates of Oxford Nanopore long reads are greater compared to Illumina short reads. Long-read assemblers using a variety of assembly algorithms have been developed to overcome this deficiency, which have not been benchmarked for genomic analyses of bacterial pathogens using Oxford Nanopore long reads. In this study, long-read assemblers, namely Canu, Flye, Miniasm/Racon, Raven, Redbean, and Shasta, were thus benchmarked using Oxford Nanopore long reads of bacterial pathogens. Ten species were tested for mediocre- and low-quality simulated reads, and 10 species were tested for real reads. Raven was the most robust assembler, obtaining complete and accurate genomes. All Miniasm/Racon and Raven assemblies of mediocre-quality reads provided accurate antimicrobial resistance (AMR) profiles, while the Raven assembly of Klebsiella variicola with low-quality reads was the only assembly with an accurate AMR profile among all assemblers and species. All assemblers functioned well for predicting virulence genes using mediocre-quality and real reads, whereas only the Raven assemblies of low-quality reads had accurate numbers of virulence genes. Regarding multilocus sequence typing (MLST), Miniasm/Racon was the most effective assembler for mediocre-quality reads, while only the Raven assemblies of Escherichia coli O157:H7 and K. variicola with low-quality reads showed positive MLST results. Miniasm/Racon and Raven were the best performers for MLST using real reads. The Miniasm/Racon and Raven assemblies showed accurate phylogenetic inference. For the pan-genome analyses, Raven was the strongest assembler for simulated reads, whereas Miniasm/Racon and Raven performed the best for real reads. Overall, the most robust and accurate assembler was Raven, closely followed by Miniasm/Racon.

scholarly journals Trycycler: consensus long-read assemblies for bacterial genomes

2021 ◽  
Author(s):  
Ryan R Wick ◽  
Louise M Judd ◽  
Louise T Cerdeira ◽  
Jane Hawkey ◽  
Guillaume Meric ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Bacterial Genomes ◽  
Manual Intervention ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Multiple Input ◽  
Long Read ◽  
Complete Bacterial Genome ◽  
Genome Assemblies ◽  
Reference Genomes

Assembly of bacterial genomes from long-read data (generated by Oxford Nanopore or Pacific Biosciences platforms) can often be complete: a single contig for each chromosome or plasmid in the genome. However, even complete bacterial genome assemblies constructed solely from long reads still contain a variety of errors, and different assemblies of the same genome often contain different errors. Here, we present Trycycler, a tool which produces a consensus assembly from multiple input assemblies of the same genome. Benchmarking using both simulated and real sequencing reads showed that Trycycler consensus assemblies contained fewer errors than any of those constructed with a single long-read assembler. Post-assembly polishing with Medaka and Pilon further reduced errors and yielded the most accurate genome assemblies in our study. As Trycycler can require human judgement and manual intervention, its output is not deterministic, and different users can produce different Trycycler assemblies from the same input data. However, we demonstrated that multiple users with minimal training converge on similar assemblies that are consistently more accurate than those produced by automated assembly tools. We therefore recommend Trycycler+Medaka+Pilon as an ideal approach for generating high-quality bacterial reference genomes.

scholarly journals Long read sequencing of 3,622 Icelanders provides insight into the role of structural variants in human diseases and other traits

2019 ◽  
Author(s):  
Doruk Beyter ◽  
Helga Ingimundardottir ◽  
Asmundur Oddsson ◽  
Hannes P. Eggertsson ◽  
Eythor Bjornsson ◽  
...  
Keyword(s):  
Sequence Data ◽  
P Value ◽  
Repeat Region ◽  
Structural Variants ◽  
Genome Wide ◽  
Population Average ◽  
Long Reads ◽  
Oxford Nanopore ◽  
A Genome ◽  
Long Read

Long-read sequencing (LRS) promises to improve characterization of structural variants (SVs), a major source of genetic diversity. We generated LRS data on 3,622 Icelanders using Oxford Nanopore Technologies, and identified a median of 22,636 SVs per individual (a median of 13,353 insertions and 9,474 deletions), spanning a median of 10 Mb per haploid genome. We discovered a set of 133,886 reliably genotyped SV alleles and imputed them into 166,281 individuals to explore their effects on diseases and other traits. We discovered an association with a rare (AF = 0.037%) deletion of the first exon of PCSK9. Carriers of this deletion have 0.93 mmol/L (1.31 SD) lower LDL cholesterol levels than the population average (p-value = 7.0·10−20). We also discovered an association with a multi-allelic SV inside a large repeat region, contained within single long reads, in an exon of ACAN. Within this repeat region we found 11 alleles that differ in the number of a 57 bp-motif repeat, and observed a linear relationship (0.016 SD per motif inserted, p = 6.2·10−18) between the number of repeats carried and height. These results show that SVs can be accurately characterized at population scale using long read sequence data in a genome-wide non-targeted approach and demonstrate how SVs impact phenotypes.

scholarly journals De-novo chromosome level assembly of plant genomes from long read sequence data

2021 ◽  
Author(s):  
Priyanka Sharma ◽  
Ardashir Kharabian Masouleh ◽  
Bruce Topp ◽  
Agnelo Furtado ◽  
Robert J. Henry
Keyword(s):  
De Novo ◽  
Sequence Data ◽  
Genetic Maps ◽  
Sequence Contigs ◽  
Plant Genomes ◽  
Proximity Analysis ◽  
Long Reads ◽  
A Genome ◽  
Long Read ◽  
Chromosome Level

SummaryRecent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. The chromosome level assembly of the contigs generated from long read sequencing has involved the use of proximity analysis (Hi-C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (PacBio HiFi) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii. a genome that has been used as model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig and the other 6 assembled into 2-4 main contigs. The small number of chromosome breaks appear to be due to highly repetitive regions of ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now seems possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed.Significance statement (of up to two sentences)De novo assembly of near complete chromosome level plant genomes is now possible using current long read sequencing and assembly tools.

scholarly journals Long-read assembly of the Brassica napus reference genome Darmor-bzh

GigaScience ◽  
2020 ◽  
Vol 9 (12) ◽  
Author(s):  
Mathieu Rousseau-Gueutin ◽  
Caroline Belser ◽  
Corinne Da Silva ◽  
Gautier Richard ◽  
Benjamin Istace ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Reference Genome ◽  
Crop Improvement ◽  
Genetic Maps ◽  
Sequencing Data ◽  
Long Reads ◽  
Gene Catalogue ◽  
Long Read ◽  
Genome Assemblies ◽  
Selection Of

Abstract Background The combination of long reads and long-range information to produce genome assemblies is now accepted as a common standard. This strategy not only allows access to the gene catalogue of a given species but also reveals the architecture and organization of chromosomes, including complex regions such as telomeres and centromeres. The Brassica genus is not exempt, and many assemblies based on long reads are now available. The reference genome for Brassica napus, Darmor-bzh, which was published in 2014, was produced using short reads and its contiguity was extremely low compared with current assemblies of the Brassica genus. Findings Herein, we report the new long-read assembly of Darmor-bzh genome (Brassica napus) generated by combining long-read sequencing data and optical and genetic maps. Using the PromethION device and 6 flowcells, we generated ∼16 million long reads representing 93× coverage and, more importantly, 6× with reads longer than 100 kb. This ultralong-read dataset allows us to generate one of the most contiguous and complete assemblies of a Brassica genome to date (contig N50 > 10 Mb). In addition, we exploited all the advantages of the nanopore technology to detect modified bases and sequence transcriptomic data using direct RNA to annotate the genome and focus on resistance genes. Conclusion Using these cutting-edge technologies, and in particular by relying on all the advantages of the nanopore technology, we provide the most contiguous Brassica napus assembly, a resource that will be valuable to the Brassica community for crop improvement and will facilitate the rapid selection of agronomically important traits.

scholarly journals High resolution species detection: accurate long read eDNA metabarcoding of North Sea fish using Oxford Nanopore sequencing

2021 ◽  
Author(s):  
Karlijn Doorenspleet ◽  
Lara Jansen ◽  
Saskia Oosterbroek ◽  
Oscar Bos ◽  
Pauline Kamermans ◽  
...  
Keyword(s):  
High Resolution ◽  
North Sea ◽  
Fish Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Nanopore Sequencing ◽  
Nature Restoration ◽  
Oxford Nanopore ◽  
Field Samples ◽  
Long Read

To monitor the effect of nature restoration projects in North Sea ecosystems, accurate and intensive biodiversity assessments are vital. DNA based techniques and especially environmental DNA (eDNA) metabarcoding from seawater is becoming a powerful monitoring tool. However, current approaches are based on genetic target regions of <500 nucleotides, which offer limited taxonomic resolution. This study aims to develop and validate a long read nanopore sequencing method for eDNA that enables improved identification of fish species. We designed a universal primer pair targeting a 2kb region covering the 12S and 16S rRNA genes of fish mitochondria. eDNA was amplified and sequenced using the Oxford Nanopore MiniON. Sequence data was processed using the new pipeline Decona, and accurate consensus identities of above 99.9% were retrieved. The primer set efficiency was tested with eDNA from a 3.000.000 L zoo aquarium with 31 species of bony fish and elasmobranchs. Over 55% of the species present were identified on species level and over 75% on genus level. Next, our long read eDNA metabarcoding approach was applied to North Sea eDNA field samples collected at ship wreck sites, the Gemini Offshore Wind Farm, the Borkum Reef Grounds and a bare sand bottom. Here, location specific fish and vertebrate communities were obtained. Incomplete reference databases still form a major bottleneck in further developing high resolution long read metabarcoding. Yet, the method has great potential for rapid and accurate fish species monitoring in marine field studies.

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