scholarly journals Parameter exploration improves the accuracy of long-read genome assembly

2021 ◽  
Author(s):  
Anurag Priyam ◽  
Alicja Witwicka ◽  
Anindita Brahma ◽  
Eckart Stolle ◽  
Yannick Wurm
Keyword(s):  
Genome Assembly ◽  
Reference Genome ◽  
Error Rates ◽  
Fine Tuning ◽  
Sequencing Error ◽  
High Quality ◽  
Long Read ◽  
Genome Assemblies ◽  
Error Profiles

Long-molecule sequencing is now routinely applied to generate high-quality reference genome assemblies. However, datasets differ in repeat composition, heterozygosity, read lengths and error profiles. The assembly parameters that provide the best results could thus differ across datasets. By integrating four complementary and biologically meaningful metrics, we show that simple fine-tuning of assembly parameters can substantially improve the quality of long-read genome assemblies. In particular, modifying estimates of sequencing error rates improves some metrics more than two-fold. We provide a flexible software, CompareGenomeQualities, that automates comparisons of assembly qualities for researchers wanting a straightforward mechanism for choosing among multiple assemblies.

scholarly journals dnAQET: a framework to compute a consolidated metric for benchmarking quality of de novo assemblies

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Gokhan Yavas ◽  
Huixiao Hong ◽  
Wenming Xiao
Keyword(s):  
Quality Assessment ◽  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Quality Score ◽  
De Novo Genome Assembly ◽  
Genome Assemblies ◽  
Reference Genomes ◽  
Better Than

Abstract Background Accurate de novo genome assembly has become reality with the advancements in sequencing technology. With the ever-increasing number of de novo genome assembly tools, assessing the quality of assemblies has become of great importance in genome research. Although many quality metrics have been proposed and software tools for calculating those metrics have been developed, the existing tools do not produce a unified measure to reflect the overall quality of an assembly. Results To address this issue, we developed the de novo Assembly Quality Evaluation Tool (dnAQET) that generates a unified metric for benchmarking the quality assessment of assemblies. Our framework first calculates individual quality scores for the scaffolds/contigs of an assembly by aligning them to a reference genome. Next, it computes a quality score for the assembly using its overall reference genome coverage, the quality score distribution of its scaffolds and the redundancy identified in it. Using synthetic assemblies randomly generated from the latest human genome build, various builds of the reference genomes for five organisms and six de novo assemblies for sample NA24385, we tested dnAQET to assess its capability for benchmarking quality evaluation of genome assemblies. For synthetic data, our quality score increased with decreasing number of misassemblies and redundancy and increasing average contig length and coverage, as expected. For genome builds, dnAQET quality score calculated for a more recent reference genome was better than the score for an older version. To compare with some of the most frequently used measures, 13 other quality measures were calculated. The quality score from dnAQET was found to be better than all other measures in terms of consistency with the known quality of the reference genomes, indicating that dnAQET is reliable for benchmarking quality assessment of de novo genome assemblies. Conclusions The dnAQET is a scalable framework designed to evaluate a de novo genome assembly based on the aggregated quality of its scaffolds (or contigs). Our results demonstrated that dnAQET quality score is reliable for benchmarking quality assessment of genome assemblies. The dnQAET can help researchers to identify the most suitable assembly tools and to select high quality assemblies generated.

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.

scholarly journals RefKA: A fast and efficient long-read genome assembly approach for large and complex genomes

2020 ◽  
Author(s):  
Yuxuan Yuan ◽  
Philipp E. Bayer ◽  
Robyn Anderson ◽  
HueyTyng Lee ◽  
Chon-Kit Kenneth Chan ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Chinese Spring ◽  
Complete Genome ◽  
Reference Genome ◽  
Computing Time ◽  
Link Type ◽  
Recent Advances ◽  
Long Read ◽  
Genome Assemblies

AbstractRecent advances in long-read sequencing have the potential to produce more complete genome assemblies using sequence reads which can span repetitive regions. However, overlap based assembly methods routinely used for this data require significant computing time and resources. Here, we have developed RefKA, a reference-based approach for long read genome assembly. This approach relies on breaking up a closely related reference genome into bins, aligning k-mers unique to each bin with PacBio reads, and then assembling each bin in parallel followed by a final bin-stitching step. During benchmarking, we assembled the wheat Chinese Spring (CS) genome using publicly available PacBio reads in parallel in 168 wall hours on a 250 CPU system. The maximum RAM used was 300 Gb and the computing time was 42,000 CPU hours. The approach opens applications for the assembly of other large and complex genomes with much-reduced computing requirements. The RefKA pipeline is available at https://github.com/AppliedBioinformatics/RefKA

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 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 Long-read amplicon denoising

2018 ◽  
Author(s):  
Venkatesh Kumar ◽  
Thomas Vollbrecht ◽  
Mark Chernyshev ◽  
Sanjay Mohan ◽  
Brian Hanst ◽  
...  
Keyword(s):  
Ground Truth ◽  
Amplicon Sequencing ◽  
Error Rates ◽  
Sequencing Error ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Medium Length ◽  
Long Reads ◽  
Long Read ◽  
Error Profiles

Long-read next generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies. Called “amplicon denoising”, this problem has been extensively studied for short-read sequencing technologies, but current solutions do not appear to generalize well to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads (here ~2.6kb) and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower.On one real dataset with ground truth, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method.Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD) are implemented purely in the Julia scientific computing language, and are hereby released along with a complete toolkit of functions that allow long-read amplicon sequence analysis pipelines to be constructed in pure Julia. Further, we make available a webserver to dramatically simplify the processing of long-read PacBio sequences.

42 An Improved, High-quality Ovine Reference Genome Assembly

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 23-24
Author(s):  
Kimberly M Davenport ◽  
Derek M Bickhart ◽  
Kim Worley ◽  
Shwetha C Murali ◽  
Noelle Cockett ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Functional Annotation ◽  
Reference Genome ◽  
De Novo ◽  
The United States ◽  
Read Length ◽  
Chromosome 11 ◽  
High Quality ◽  
Oxford Nanopore ◽  
Long Read

Abstract Sheep are an important agricultural species used for both food and fiber in the United States and globally. A high-quality reference genome enhances the ability to discover genetic and biological mechanisms influencing important traits, such as meat and wool quality. The rapid advances in genome assembly algorithms and emergence of increasingly long sequence read length provide the opportunity for an improved de novo assembly of the sheep reference genome. Tissue was collected postmortem from an adult Rambouillet ewe selected by USDA-ARS for the Ovine Functional Annotation of Animal Genomes project. Short-read (55x coverage), long-read PacBio (75x coverage), and Hi-C data from this ewe were retrieved from public databases. We generated an additional 50x coverage of Oxford Nanopore data and assembled the combined long-read data with canu v1.9. The assembled contigs were polished with Nanopolish v0.12.5 and scaffolded using Hi-C data with Salsa v2.2. Gaps were filled with PBsuite v15.8.24 and polished with Nanopolish v0.12.5 followed by removal of duplicate contigs with PurgeDups v1.0.1. Chromosomes were oriented by identifying centromeres and telomeres with RepeatMasker v4.1.1, indicating a need to reverse the orientation of chromosome 11 relative to Oar_rambouillet_v1.0. Final polishing was performed with two rounds of a pipeline which consisted of freebayes v1.3.1 to call variants, Merfin to validate them, and BCFtools to generate the consensus fasta. The ARS-UI_Ramb_v2.0 assembly has improved continuity (contig N50 of 43.19 Mb) with a 19-fold and 38-fold decrease in the number of scaffolds compared with Oar_rambouillet_v1.0 and Oar_v4.0. ARS-UI_Ramb_v2.0 has greater per-base accuracy and fewer insertions and deletions identified from mapped RNA sequence than previous assemblies. This significantly improved reference assembly, public at NCBI GenBank under accession number GCA_016772045, will optimize the functional annotation of the sheep genome and facilitate improved mapping accuracy of genetic variant and expression data for traits relevant the sheep industry.

scholarly journals A high-quality de novo genome assembly based on nanopore sequencing of a wild-caught coconut rhinoceros beetle (Oryctes rhinoceros)

2021 ◽  
Author(s):  
Igor Filipović ◽  
Gordana Rašić ◽  
James Hereward ◽  
Maria Gharuka ◽  
Gregor J Devine ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Nuclear Genome ◽  
Assembly Process ◽  
Structural Annotation ◽  
High Quality ◽  
Oryctes Rhinoceros ◽  
Rhinoceros Beetle ◽  
Long Read ◽  
Genome Assemblies

Background: An optimal starting point for relating genome function to organismal biology is a high-quality nuclear genome assembly, and long-read sequencing is revolutionizing the production of this genomic resource in insects. Despite this, nuclear genome assemblies have been under-represented for agricultural insect pests, particularly from the order Coleoptera. Here we present a de novo genome assembly and structural annotation for the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), based on Oxford Nanopore Technologies (ONT) long-read data generated from a wild-caught female, as well as the assembly process that also led to the recovery of the complete circular genome assemblies of the beetle's mitochondrial genome and that of the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). As an invasive pest of palm trees, O. rhinoceros is undergoing an expansion in its range across the Pacific Islands, requiring new approaches to management that may include strategies facilitated by genome assembly and annotation. Results: High-quality DNA isolated from an adult female was used to create four ONT libraries that were sequenced using four MinION flow cells, producing a total of 27.2 Gb of high-quality long-read sequences. We employed an iterative assembly process and polishing with one lane of high-accuracy Illumina reads, obtaining a final size of the assembly of 377.36 Mb that had high contiguity (fragment N50 length = 12 Mb) and accuracy, as evidenced by the exceptionally high completeness of the benchmarked set of conserved single-copy orthologous genes (BUSCO completeness = 99.11%). These quality metrics place our assembly as the most complete of the published Coleopteran genomes. The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes showing BUSCO completeness of 92.09%, as well as the expected number of non-coding RNAs and the number and structure of paralogous genes in a gene family like Sigma GST. Conclusions: The genomic resources produced in this study form a foundation for further functional genetic research and management programs that may inform the control and surveillance of O. rhinoceros populations, and we demonstrate the efficacy of de novo genome assembly using long-read ONT data from a single field-caught insect.

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.

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