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 Hapo-G, Haplotype-Aware Polishing Of Genome Assemblies

2020 ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
Short Reads ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

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 short reads to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

scholarly journals Long-read and chromosome-scale assembly of the hexaploid wheat genome achieves higher resolution for research and breeding

2021 ◽  
Author(s):  
Jean-Marc Aury ◽  
Stefan Engelen ◽  
Benjamin Istace ◽  
Cécile Monat ◽  
Pauline Lasserre-Zuber ◽  
...  
Keyword(s):  
Bread Wheat ◽  
Rapid Evolution ◽  
Wheat Genome ◽  
High Quality ◽  
Sequencing Technologies ◽  
Repeat Content ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Reference Quality

AbstractThe sequencing of the wheat (Triticum aestivum) genome has been a methodological challenge for many years due to its large size (15.5 Gb), repeat content, and hexaploidy. Many initiatives aiming at obtaining a reference genome of cultivar Chinese Spring have been launched in the past years and it was achieved in 2018 as the result of a huge effort to combine short-read whole genome sequencing with many other resources. Reference-quality genome assemblies were then produced for other accessions but the rapid evolution of sequencing technologies offers opportunities to reach high-quality standards at lower cost. Here, we report on an optimized procedure based on long-reads produced on the ONT (Oxford Nanopore Technology) PromethION device to assemble the genome of the French bread wheat cultivar Renan. We provide the most contiguous and complete chromosome-scale assembly of a bread wheat genome to date, a resource that will be valuable for the crop community and will facilitate the rapid selection of agronomically important traits. We also provide the methodological standards to generate high-quality assemblies of complex genomes.

scholarly journals Highly contiguous assemblies of 101 drosophilid genomes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Bernard Y Kim ◽  
Jeremy Wang ◽  
Danny E Miller ◽  
Olga Barmina ◽  
Emily Kay Delaney ◽  
...  
Keyword(s):  
High Quality ◽  
Coding Regions ◽  
Public Resource ◽  
Oxford Nanopore ◽  
Starting Point ◽  
Long Read ◽  
Species Groups ◽  
Genome Assemblies ◽  
High Quality Genome ◽  
Laboratory Protocol

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

scholarly journals WENGAN: Efficient and high quality hybrid de novo assembly of human genomes

2019 ◽  
Author(s):  
Alex Di Genova ◽  
Elena Buena-Atienza ◽  
Stephan Ossowski ◽  
Marie-France Sagot
Keyword(s):  
De Novo ◽  
Computational Cost ◽  
Sequence Information ◽  
Sequencing Data ◽  
High Quality ◽  
Sequencing Technologies ◽  
Human Genomes ◽  
Long Reads ◽  
Long Read ◽  
Genome Assemblies

The continuous improvement of long-read sequencing technologies along with the development of ad-doc algorithms has launched a new de novo assembly era that promises high-quality genomes. However, it has proven difficult to use only long reads to generate accurate genome assemblies of large, repeat-rich human genomes. To date, most of the human genomes assembled from long error-prone reads add accurate short reads to further polish the consensus quality. Here, we report the development of a novel algorithm for hybrid assembly, WENGAN, and the de novo assembly of four human genomes using a combination of sequencing data generated on ONT PromethION, PacBio Sequel, Illumina and MGI technology. WENGAN implements efficient algorithms that exploit the sequence information of short and long reads to tackle assembly contiguity as well as consensus quality. The resulting genome assemblies have high contiguity (contig NG50:16.67-62.06 Mb), few assembly errors (contig NGA50:10.9-45.91 Mb), good consensus quality (QV:27.79-33.61), and high gene completeness (BUSCO complete: 94.6-95.1%), while consuming low computational resources (CPU hours:153-1027). In particular, the WENGAN assembly of the haploid CHM13 sample achieved a contig NG50 of 62.06 Mb (NGA50:45.91 Mb), which surpasses the contiguity of the current human reference genome (GRCh38 contig NG50:57.88 Mb). Providing highest quality at low computational cost, WENGAN is an important step towards the democratization of the de novo assembly of human genomes. The WENGAN assembler is available at https://github.com/adigenova/wengan

scholarly journals GENOME REPORT: High-quality genome assemblies of 15 Drosophila species generated using Nanopore sequencing

2018 ◽  
Author(s):  
Danny E. Miller ◽  
Cynthia Staber ◽  
Julia Zeitlinger ◽  
R. Scott Hawley
Keyword(s):  
Single Molecule ◽  
Drosophila Species ◽  
High Quality ◽  
Additional Species ◽  
Oxford Nanopore ◽  
Wide Range ◽  
Long Read ◽  
Genome Assemblies ◽  
High Quality Genome ◽  
Reference Genomes

ABSTRACTThe Drosophila genus is a unique group containing a wide range of species that occupy diverse ecosystems. In addition to the most widely studied species, Drosophila melanogaster, many other members in this genus also possess a well-developed set of genetic tools. Indeed, high-quality genomes exist for several species within the genus, facilitating studies of the function and evolution of cis-regulatory regions and proteins by allowing comparisons across at least 50 million years of evolution. Yet, the available genomes still fail to capture much of the substantial genetic diversity within the Drosophila genus. We have therefore tested protocols to rapidly and inexpensively sequence and assemble the genome from any Drosophila species using single-molecule sequencing technology from Oxford Nanopore. Here, we use this technology to present high-quality genome assemblies of 15 Drosophila species: 10 of the 12 originally sequenced Drosophila species (ananassae, erecta, mojavensis, persimilis, pseudoobscura, sechellia, simulans, virilis, willistoni, and yakuba), four additional species that had previously reported assemblies (biarmipes, bipectinata, eugracilis, and mauritiana), and one novel assembly (triauraria). Genomes were generated from an average of 29x depth-of-coverage data that after assembly resulted in an average contig N50 of 4.4 Mb. Subsequent alignment of contigs from the published reference genomes demonstrates that our assemblies could be used to close over 60% of the gaps present in the currently published reference genomes. Importantly, the materials and reagents cost for each genome was approximately $1,000 (USD). This study demonstrates the power and cost-effectiveness of long-read sequencing for genome assembly in Drosophila and provides a framework for the affordable sequencing and assembly of additional Drosophila genomes.

scholarly journals SVIM: Structural Variant Identification using Mapped Long Reads

2018 ◽  
Author(s):  
David Heller ◽  
Martin Vingron
Keyword(s):  
Single Molecule ◽  
Simulated Data ◽  
Structural Variants ◽  
Human Phenotype ◽  
Structural Variant ◽  
Small Indels ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read

AbstractMotivationStructural variants are defined as genomic variants larger than 50bp. They have been shown to affect more bases in any given genome than SNPs or small indels. Additionally, they have great impact on human phenotype and diversity and have been linked to numerous diseases. Due to their size and association with repeats, they are difficult to detect by shotgun sequencing, especially when based on short reads. Long read, single molecule sequencing technologies like those offered by Pacific Biosciences or Oxford Nanopore Technologies produce reads with a length of several thousand base pairs. Despite the higher error rate and sequencing cost, long read sequencing offers many advantages for the detection of structural variants. Yet, available software tools still do not fully exploit the possibilities.ResultsWe present SVIM, a tool for the sensitive detection and precise characterization of structural variants from long read data. SVIM consists of three components for the collection, clustering and combination of structural variant signatures from read alignments. It discriminates five different variant classes including similar types, such as tandem and interspersed duplications and novel element insertions. SVIM is unique in its capability of extracting both the genomic origin and destination of duplications. It compares favorably with existing tools in evaluations on simulated data and real datasets from PacBio and Nanopore sequencing machines.Availability and implementationThe source code and executables of SVIM are available on Github: github.com/eldariont/svim. SVIM has been implemented in Python 3 and published on bioconda and the Python Package [email protected]

scholarly journals PhaseME: Automatic rapid assessment of phasing quality and phasing improvement

GigaScience ◽  
2020 ◽  
Vol 9 (7) ◽  
Author(s):  
Sina Majidian ◽  
Fritz J Sedlazeck
Keyword(s):  
Rapid Assessment ◽  
Linkage Data ◽  
Universal Method ◽  
High Quality ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Linkage Information ◽  
Quality Assessments

Abstract Background The detection of which mutations are occurring on the same DNA molecule is essential to predict their consequences. This can be achieved by phasing the genomic variations. Nevertheless, state-of-the-art haplotype phasing is currently a black box in which the accuracy and quality of the reconstructed haplotypes are hard to assess. Findings Here we present PhaseME, a versatile method to provide insights into and improvement of sample phasing results based on linkage data. We showcase the performance and the importance of PhaseME by comparing phasing information obtained from Pacific Biosciences including both continuous long reads and high-quality consensus reads, Oxford Nanopore Technologies, 10x Genomics, and Illumina sequencing technologies. We found that 10x Genomics and Oxford Nanopore phasing can be significantly improved while retaining a high N50 and completeness of phase blocks. PhaseME generates reports and summary plots to provide insights into phasing performance and correctness. We observed unique phasing issues for each of the sequencing technologies, highlighting the necessity of quality assessments. PhaseME is able to decrease the Hamming error rate significantly by 22.4% on average across all 5 technologies. Additionally, a significant improvement is obtained in the reduction of long switch errors. Especially for high-quality consensus reads, the improvement is 54.6% in return for only a 5% decrease in phase block N50 length. Conclusions PhaseME is a universal method to assess the phasing quality and accuracy and improves the quality of phasing using linkage information. The package is freely available at https://github.com/smajidian/phaseme.

scholarly journals Improving the Chromosome-Level Genome Assembly of the Siamese Fighting Fish (Betta splendens) in a University Master’s Course

2020 ◽  
Vol 10 (7) ◽  
pp. 2179-2183 ◽  
Author(s):  
Stefan Prost ◽  
Malte Petersen ◽  
Martin Grethlein ◽  
Sarah Joy Hahn ◽  
Nina Kuschik-Maczollek ◽  
...  
Keyword(s):  
Genome Assembly ◽  
High Throughput Sequencing ◽  
Siamese Fighting Fish ◽  
Betta Splendens ◽  
High Quality ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Chromosome Level

Ever decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish (Betta splendens) that was carried out during a practical university master’s course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behavior. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published Hi-C data. The use of ∼35x nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using the Hi-C data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 96.1% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly. We present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university master’s course. The use of ∼35× long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.

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 Highly contiguous assemblies of 101 drosophilid genomes

2020 ◽  
Author(s):  
Bernard Y Kim ◽  
Jeremy Wang ◽  
Danny E. Miller ◽  
Olga Barmina ◽  
Emily K. Delaney ◽  
...  
Keyword(s):  
Community Resource ◽  
High Quality ◽  
Public Resource ◽  
Oxford Nanopore ◽  
Starting Point ◽  
Long Read ◽  
Wet Lab ◽  
Species Groups ◽  
Genome Assemblies ◽  
High Quality Genome

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long read sequencing allow high quality genome assemblies for tens or even hundreds of species to be generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of high-quality assemblies for 101 lines of 95 drosophilid species encompassing 14 species groups and 35 sub-groups with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. These assemblies, along with detailed wet lab protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution within this key group.

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