The changing face of genome assemblies: guidance on achieving high‐quality reference genomes

Despite the substantial economic impact of Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) on legume productions worldwide, the genetic basis of its pathogenicity and potential host association is poorly understood. The production of high-quality reference genome assemblies of Cff strains associated with different hosts sheds light on the genetic basis of its pathogenic variability and host association. Moreover, the study of recent outbreaks of bacterial wilt and microevolution of the pathogen in Australia requires access to high-quality, reference genomes that are sufficiently closely related to the population being studied within Australia. We provide the first genome assemblies of Cff strains associated with mungbean and soybean, which revealed high variability in their plasmid composition. The analysis of Cff genomes revealed an extensive suite of carbohydrate-active enzymes potentially associated with pathogenicity, including four carbohydrate esterases, 50 glycoside hydrolases, 23 glycosyl transferases, and a polysaccharide lyase. We also identified 11 serine peptidases, three of which were located within a linear plasmid, pCff119. These high-quality assemblies and annotations will provide a foundation for population genomics studies of Cff in Australia and for answering fundamental questions regarding pathogenicity factors and adaptation of Cff to various hosts worldwide, and, at a broader scale, contribute to unravelling genomic features of Gram-positive, xylem-inhabiting bacterial pathogens.

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Complete Genome Assemblies of Three Highly Prevalent, Toxigenic Clostridioides difficile Strains Causing Health Care-Associated Infections in Australia

Microbiology Resource Announcements ◽

10.1128/mra.00599-21 ◽

2021 ◽

Vol 10 (31) ◽

Author(s):

Keeley O’Grady ◽

Thomas V. Riley ◽

Daniel R. Knight

Keyword(s):

Health Care ◽

Gastrointestinal Illness ◽

High Quality ◽

Content Type ◽

Clostridioides Difficile ◽

Hybrid Genome ◽

Life Threatening ◽

Health Care Associated Infections ◽

Genome Assemblies ◽

Reference Genomes

Clostridioides difficile infection (CDI) is the leading cause of life-threatening health care-related gastrointestinal illness worldwide. Phylogenetically appropriate closed reference genomes are essential for studies of C. difficile transmission and evolution. Here, we provide high-quality complete hybrid genome assemblies for the three most prevalent C. difficile strains causing CDI in Australia.

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

Nature ◽

10.1038/s41586-021-03451-0 ◽

2021 ◽

Vol 592 (7856) ◽

pp. 737-746 ◽

Cited By ~ 1

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.

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

10.1101/2020.05.22.110833 ◽

2020 ◽

Cited By ~ 16

Author(s):

Arang Rhie ◽

Shane A. McCarthy ◽

Olivier Fedrigo ◽

Joana Damas ◽

Giulio Formenti ◽

...

Keyword(s):

Cost Effective ◽

Lessons Learned ◽

Vertebrate Species ◽

High Quality ◽

New Era ◽

Sequencing Technologies ◽

Long Read ◽

Cartilaginous Fishes ◽

Genome Assemblies ◽

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 only available for 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 the most accurate and complete reference genomes to date. Here we summarize these developments, introduce a set of quality standards, and present lessons learned from sequencing and assembling 16 species representing major vertebrate lineages (mammals, birds, reptiles, amphibians, teleost fishes and cartilaginous fishes). 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 error in assemblies. Our new assemblies identify and correct substantial errors in some of the best historical reference genomes. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an effort to generate high-quality, complete reference genomes for all ~70,000 extant vertebrate species and help enable a new era of discovery across the life sciences.

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

10.1101/267393 ◽

2018 ◽

Cited By ~ 6

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.

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Automated assembly scaffolding elevates a new tomato system for high-throughput genome editing

10.1101/2021.11.18.469135 ◽

2021 ◽

Author(s):

Michael Alonge ◽

Ludivine Lebeigle ◽

Melanie Kirsche ◽

Sergey Aganezov ◽

Xingang Wang ◽

...

Keyword(s):

Functional Genomics ◽

Genome Editing ◽

High Throughput ◽

Rapid Cycling ◽

Automated Assembly ◽

High Quality ◽

Genetic Systems ◽

Genome Assemblies ◽

Tomato Genotype ◽

Reference Genomes

Advancing crop genomics requires efficient genetic systems enabled by high-quality personalized genome assemblies. Here, we introduce RagTag, a toolset for automating assembly scaffolding and patching, and we establish chromosome-scale reference genomes for the widely used tomato genotype M82 along with Sweet-100, a rapid-cycling genotype that we developed to accelerate functional genomics and genome editing. This work outlines strategies to rapidly expand genetic systems and genomic resources in other plant species.

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

NAR Genomics and Bioinformatics ◽

10.1093/nargab/lqab034 ◽

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.

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Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird-of-paradise

10.1101/2019.12.19.882399 ◽

2019 ◽

Cited By ~ 5

Author(s):

Valentina Peona ◽

Mozes P.K. Blom ◽

Luohao Xu ◽

Reto Burri ◽

Shawn Sullivan ◽

...

Keyword(s):

Dark Matter ◽

Genome Assembly ◽

Sex Chromosome ◽

De Novo ◽

Model Organism ◽

Technology Choice ◽

High Quality ◽

Sequencing Technologies ◽

Downstream Analysis ◽

Genome Assemblies

AbstractGenome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies have opened up a whole new world of genomic biodiversity. Although these technologies generate high-quality genome assemblies, there are still genomic regions difficult to assemble, like repetitive elements and GC-rich regions (genomic “dark matter”). In this study, we compare the efficiency of currently used sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter starting from the same sample. By adopting different de-novo assembly strategies, we were able to compare each individual draft assembly to a curated multiplatform one and identify the nature of the previously missing dark matter with a particular focus on transposable elements, multi-copy MHC genes, and GC-rich regions. Thanks to this multiplatform approach, we demonstrate the feasibility of producing a high-quality chromosome-level assembly for a non-model organism (paradise crow) for which only suboptimal samples are available. Our approach was able to reconstruct complex chromosomes like the repeat-rich W sex chromosome and several GC-rich microchromosomes. Telomere-to-telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects around the completeness of both the coding and non-coding parts of the genomes.

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Population-scale long-read sequencing uncovers transposable elements contributing to gene expression variation and associated with adaptive signatures in Drosophila melanogaster

10.1101/2021.10.08.463646 ◽

2021 ◽

Author(s):

Gabriel Rech ◽

Santiago Radio ◽

Sara Guirao-Rico ◽

Laura Aguilera ◽

Vivien Horvath ◽

...

Keyword(s):

Gene Expression ◽

Genetic Variation ◽

Transposable Elements ◽

Natural Populations ◽

Gene Expression Variation ◽

High Quality ◽

Expression Variation ◽

Climatic Regions ◽

Genomic Studies ◽

Reference Genomes

High quality reference genomes are crucial to understanding genome function, structure and evolution. The availability of reference genomes has allowed us to start inferring the role of genetic variation in biology, disease, and biodiversity conservation. However, analyses across organisms demonstrate that a single reference genome is not enough to capture the global genetic diversity present in populations. In this work, we generated 32 high-quality reference genomes for the well-known model species D. melanogaster and focused on the identification and analysis of transposable element variation as they are the most common type of structural variant. We showed that integrating the genetic variation across natural populations from five climatic regions increases the number of detected insertions by 58%. Moreover, 26% to 57% of the insertions identified using long-reads were missed by short-reads methods. We also identified hundreds of transposable elements associated with gene expression variation and new TE variants likely to contribute to adaptive evolution in this species. Our results highlight the importance of incorporating the genetic variation present in natural populations to genomic studies, which is essential if we are to understand how genomes function and evolve.

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SIGAR: Inferring features of genome architecture and DNA rearrangements by split read mapping

10.1101/2020.05.05.079426 ◽

2020 ◽

Author(s):

Yi Feng ◽

Leslie Y. Beh ◽

Wei-Jen Chang ◽

Laura F. Landweber

Keyword(s):

Genome Assembly ◽

Repetitive Sequences ◽

Genome Architecture ◽

Dna Rearrangements ◽

High Quality ◽

Microbial Eukaryotes ◽

Ciliate Species ◽

Split Read ◽

High Level ◽

Genome Assemblies

AbstractCiliates are microbial eukaryotes with distinct somatic and germline genomes. Post-zygotic development involves extensive remodeling of the germline genome to form somatic chromosomes. Ciliates therefore offer a valuable model for studying the architecture and evolution of programmed genome rearrangements. Current studies usually focus on a few model species, where rearrangement features are annotated by aligning reference germline and somatic genomes. While many high-quality somatic genomes have been assembled, a high quality germline genome assembly is difficult to obtain due to its smaller DNA content and abundance of repetitive sequences. To overcome these hurdles, we propose a new pipeline SIGAR (Splitread Inference of Genome Architecture and Rearrangements) to infer germline genome architecture and rearrangement features without a germline genome assembly, requiring only short germline DNA sequencing reads. As a proof of principle, 93% of rearrangement junctions identified by SIGAR in the ciliate Oxytricha trifallax were validated by the existing germline assembly. We then applied SIGAR to six diverse ciliate species without germline genome assemblies, including Ichthyophthirius multifilii, a fish pathogen. Despite the high level of somatic DNA contamination in each sample, SIGAR successfully inferred rearrangement junctions, short eliminated sequences and potential scrambled genes in each species. This pipeline enables pilot surveys or exploration of DNA rearrangements in species with limited DNA material access, thereby providing new insights into the evolution of chromosome rearrangements.

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