scholarly journals TT-Mars: Structural Variants Assessment Based on Haplotype-resolved Assemblies

2021 ◽  
Author(s):  
Jianzhi Yang ◽  
Mark Chaisson
Keyword(s):  
Gold Standard ◽  
Method Development ◽  
Point Estimate ◽  
Personal Genome ◽  
Structural Variants ◽  
High Quality ◽  
Alternative Approach ◽  
Long Read ◽  
Genome Assemblies ◽  
Validation Tool

AbstractVariant benchmarking is a critical component of method development and evaluating the accuracy of studies of genetic variation. Currently, the best approach to evaluate the accuracy of a callset is the comparison against a well curated gold standard. In repetitive regions of the genome it may be difficult to establish what is the truth for a call, for example when different alignment scoring metrics provide equally supported but different variant calls in on the same data. Here we provide an alternative approach, TT-Mars, that takes advantage of the recent production of high-quality haplotype-resolved genome assemblies by evaluating variant calls based on how well their call reflects the content of the assembly, rather than comparing calls themselves. We used TT-Mars to assess callsets from different SV discovery methods on multiple human genome samples and demonstrated that it is capable at accurately classifying true positive and false positive SVs. On the HG002 personal genome, TT-Mars recapitulates 96.0%-99.6% of the validations made using the Genome in a Bottle gold standard callset evaluated by truvari, and evaluates an additional 121-10,966 variants across different callsets. Furthermore, with a group of high-quality assemblies, TT-Mars can evaluate performance of SV calling algorithms as a distribution rather than a point estimate. We also compare TT-Mars against the long-read based validation tool, VaPoR, and when assembly-based variant calls produced by dipcall are used as a gold standard. Compared with VaPoR, TT-Mars analyzes more calls on a long read callset by assessing more short variant calls (< 100 bases), while requiring smaller input. Compared with validation using dipcall variants, TT-Mars analyzes 1,497-2,229 more calls on long read callsets and has favorable results when candidate calls are fragmented into multiple calls in alignments. TT-Mars is available at https://github.com/ChaissonLab/TT-Mars.git with accompanying assembly data and corresponding liftover files.

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 Towards a better understanding of the low recall of insertion variants with short-read based variant callers

2020 ◽  
Author(s):  
Wesley Delage ◽  
Julien Thevenon ◽  
Claire Lemaitre
Keyword(s):  
Gold Standard ◽  
Insertion Site ◽  
Structural Variants ◽  
Genomic Context ◽  
Breakpoint Junction ◽  
Short Read ◽  
Depth Analysis ◽  
Long Read ◽  
The Impact

AbstractSince 2009, numerous tools have been developed to detect structural variants (SVs) using short read technologies. Insertions >50 bp are one of the hardest type to discover and are drastically underrepresented in gold standard variant callsets. The advent of long read technologies has completely changed the situation. In 2019, two independent cross technologies studies have published the most complete variant callsets with sequence resolved insertions in human individuals. Among the reported insertions, only 17 to 37% could be discovered with short-read based tools. In this work, we performed an in-depth analysis of these unprecedented insertion callsets in order to investigate the causes of such failures. We have first established a precise classification of insertion variants according to four layers of characterization: the nature and size of the inserted sequence, the genomic context of the insertion site and the breakpoint junction complexity. Because these levels are intertwined, we then used simulations to characterize the impact of each complexity factor on the recall of several SV callers. Simulations showed that the most impacting factor was the insertion type rather than the genomic context, with various difficulties being handled differently among the tested SV callers, and they highlighted the lack of sequence resolution for most insertion calls. Our results explain the low recall by pointing out several difficulty factors among the observed insertion features and provide avenues for improving SV caller algorithms and their [email protected]

scholarly journals De novo assembly of a Tibetan genome and identification of novel structural variants associated with high-altitude adaptation

2019 ◽  
Vol 7 (2) ◽  
pp. 391-402 ◽  
Author(s):  
Yaoxi He ◽  
Haiyi Lou ◽  
Chaoying Cui ◽  
Lian Deng ◽  
Yang Gao ◽  
...  
Keyword(s):  
High Altitude ◽  
De Novo Assembly ◽  
De Novo ◽  
Population Analysis ◽  
Extreme Environments ◽  
Structural Variants ◽  
Base Pairs ◽  
High Quality ◽  
Long Read ◽  
Altitude Adaptation

Abstract Structural variants (SVs) may play important roles in human adaptation to extreme environments such as high altitude but have been under-investigated. Here, combining long-read sequencing with multiple scaffolding techniques, we assembled a high-quality Tibetan genome (ZF1), with a contig N50 length of 24.57 mega-base pairs (Mb) and a scaffold N50 length of 58.80 Mb. The ZF1 assembly filled 80 remaining N-gaps (0.25 Mb in total length) in the reference human genome (GRCh38). Markedly, we detected 17 900 SVs, among which the ZF1-specific SVs are enriched in GTPase activity that is required for activation of the hypoxic pathway. Further population analysis uncovered a 163-bp intronic deletion in the MKL1 gene showing large divergence between highland Tibetans and lowland Han Chinese. This deletion is significantly associated with lower systolic pulmonary arterial pressure, one of the key adaptive physiological traits in Tibetans. Moreover, with the use of the high-quality de novo assembly, we observed a much higher rate of genome-wide archaic hominid (Altai Neanderthal and Denisovan) shared non-reference sequences in ZF1 (1.32%–1.53%) compared to other East Asian genomes (0.70%–0.98%), reflecting a unique genomic composition of Tibetans. One such archaic hominid shared sequence—a 662-bp intronic insertion in the SCUBE2 gene—is enriched and associated with better lung function (the FEV1/FVC ratio) in Tibetans. Collectively, we generated the first high-resolution Tibetan reference genome, and the identified SVs may serve as valuable resources for future evolutionary and medical 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.

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.

scholarly journals Draft Genome Assemblies and Annotations of Agrypnia vestita Walker, and Hesperophylax magnus Banks Reveal Substantial Repetitive Element Expansion in Tube Case-making Caddisflies (Insecta: Trichoptera)

2020 ◽  
Author(s):  
Lindsey K. Olsen ◽  
Jacqueline Heckenhauer ◽  
John S. Sproul ◽  
Rebecca B. Dikow ◽  
Vanessa L. Gonzalez ◽  
...  
Keyword(s):  
Genome Size ◽  
High Potential ◽  
Genomic Diversity ◽  
High Quality ◽  
Genomic Resources ◽  
Insect Order ◽  
Genome Size Evolution ◽  
Size Evolution ◽  
Long Read ◽  
Genome Assemblies

AbstractTrichoptera (caddisflies) play an essential role in freshwater ecosystems; for instance, larvae process organic material from the water and are food for a variety of predators. Knowledge on the genomic diversity of caddisflies can facilitate comparative and phylogenetic studies thereby allowing scientists to better understand the evolutionary history of caddisflies. While Trichoptera are the most diverse aquatic insect order, they remain poorly represented in terms of genomic resources. To date, all long-read based genomes have been sequenced from individuals in the retreat-making suborder, Annulipalpia, leaving ∼275 Ma of evolution without high-quality genomic resources. Here, we report the first long-read based de novo genome assemblies of two tube case-making Trichoptera from the suborder Integripalpia, Agrypnia vestita Walker and Hesperophylax magnus Banks. We find that these tube case-making caddisflies have genome sizes that are at least three-fold larger than those of currently sequenced annulipalpian genomes and that this pattern is at least partly driven by major expansion of repetitive elements. In H. magnus, long interspersed nuclear elements (LINEs) alone exceed the entire genome size of some annulipalpian counterparts suggesting that caddisflies have high potential as a model for understanding genome size evolution in diverse insect lineages.SignificanceThere is a lack of genomic resources for aquatic insects. So far, only three high-quality genomes have been assembled, all from individuals in the retreat-making suborder Annulipalpia. In this article, we report the first high-quality genomes of two case-making species from the suborder Integripalpia, which are essential for studying genomic diversity across this ecologically diverse insect order. Our research reveals larger genome sizes in the tube case-makers (suborder Integripalpia, infraorder Phryganides), accompanied by a disproportionate increase of repetitive DNA. This suggests that genome size is at least partly driven by a major expansion of repetitive elements. Our work shows that caddisflies have high potential as a model for understanding how genomic diversity might be linked to functional diversification and forms the basis for detailed studies on genome size evolution in caddisflies.Data depositionThis project has been deposited at NCBI under the Bioproject ID: PRJNA668166

scholarly journals Towards complete and error-free genome assemblies of all vertebrate species

Nature ◽  
2021 ◽  
Vol 592 (7856) ◽  
pp. 737-746 ◽  
Author(s):  
Arang Rhie ◽  
Shane A. McCarthy ◽  
Olivier Fedrigo ◽  
Joana Damas ◽  
Giulio Formenti ◽  
...  
Keyword(s):  
Cost Effective ◽  
Lessons Learned ◽  
Vertebrate Species ◽  
High Quality ◽  
Protein Coding ◽  
Sequencing Technologies ◽  
Long Read ◽  
Genome Assemblies ◽  
Assembly Error ◽  
Reference Genomes

AbstractHigh-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1–4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

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 Comparative Annotation Toolkit (CAT) - simultaneous clade and personal genome annotation

2017 ◽  
Author(s):  
Ian T. Fiddes ◽  
Joel Armstrong ◽  
Mark Diekhans ◽  
Stefanie Nachtweide ◽  
Zev N. Kronenberg ◽  
...  
Keyword(s):  
Genome Annotation ◽  
De Novo ◽  
Low Cost ◽  
Great Apes ◽  
Personal Genome ◽  
Sequencing Technologies ◽  
Human Genomes ◽  
Long Read ◽  
Genome Assemblies ◽  
Rat Genome

ABSTRACTThe recent introductions of low-cost, long-read, and read-cloud sequencing technologies coupled with intense efforts to develop efficient algorithms have made affordable, high-quality de novo sequence assembly a realistic proposition. The result is an explosion of new, ultra-contiguous genome assemblies. To compare these genomes we need robust methods for genome annotation. We describe the fully open source Comparative Annotation Toolkit (CAT), which provides a flexible way to simultaneously annotate entire clades and identify orthology relationships. We show that CAT can be used to improve annotations on the rat genome, annotate the great apes, annotate a diverse set of mammals, and annotate personal, diploid human genomes. We demonstrate the resulting discovery of novel genes, isoforms and structural variants, even in genomes as well studied as rat and the great apes, and how these annotations improve cross-species RNA expression experiments.

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.

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