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July 19, 2019

De novo assembly of haplotype-resolved genomes with trio binning.

Complex allelic variation hampers the assembly of haplotype-resolved sequences from diploid genomes. We developed trio binning, an approach that simplifies haplotype assembly by resolving allelic variation before assembly. In contrast with prior approaches, the effectiveness of our method improved with increasing heterozygosity. Trio binning uses short reads from two parental genomes to first partition long reads from an offspring into haplotype-specific sets. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction. We used trio binning to recover both haplotypes of a diploid human genome and identified complex structural variants missed by alternative approaches. We sequenced an F1 cross between the cattle subspecies Bos taurus taurus and Bos taurus indicus and completely assembled both parental haplotypes with NG50 haplotig sizes of >20 Mb and 99.998% accuracy, surpassing the quality of current cattle reference genomes. We suggest that trio binning improves diploid genome assembly and will facilitate new studies of haplotype variation and inheritance.

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July 19, 2019

Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

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July 7, 2019

Do echinoderm genomes measure up?

Echinoderm genome sequences are a corpus of useful information about a clade of animals that serve as research models in fields ranging from marine ecology to cell and developmental biology. Genomic information from echinoids has contributed to insights into the gene interactions that drive the developmental process at the molecular level. Such insights often rely heavily on genomic information and the kinds of questions that can be asked thus depend on the quality of the sequence information. Here we describe the history of echinoderm genomic sequence assembly and present details about the quality of the data obtained. All of the sequence information discussed here is posted on the echinoderm information web system, Echinobase.org. Copyright © 2015 Elsevier B.V. All rights reserved.

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July 7, 2019

Best practices in insect genome sequencing: What works and what doesn’t.

The last decade of decreasing DNA sequencing costs and proliferating sequencing services in core labs and companies has brought the de-novo genome sequencing and assembly of insect species within reach for many entomologists. However, sequence production alone is not enough to generate a high quality reference genome, and in many cases, poor planning can lead to extremely fragmented genome assemblies preventing high quality gene annotation and other desired analyses. Insect genomes can be problematic to assemble, due to combinations of high polymorphism, inability to breed for genome homozygocity, and small physical sizes limiting the quantity of DNA able to be isolated from a single individual. Recent advances in sequencing technology and assembly strategies are enabling a revolution for insect genome reference sequencing and assembly. Here we review historical and new genome sequencing and assembly strategies, with a particular focus on their application to arthropod genomes. We highlight both the need to design sequencing strategies for the requirements of the assembly software, and new long-read technologies that are enabling a return to traditional assembly approaches. Finally, we compare and contrast very cost effective short read draft genome strategies with the long read approaches that although entailing additional cost, bring a higher likelihood of success and the possibility of archival assembly qualities approaching that of finished genomes.

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July 7, 2019

It’s more than stamp collecting: how genome sequencing can unify biological research.

The availability of reference genome sequences, especially the human reference, has revolutionized the study of biology. However, while the genomes of some species have been fully sequenced, a wide range of biological problems still cannot be effectively studied for lack of genome sequence information. Here, I identify neglected areas of biology and describe how both targeted species sequencing and more broad taxonomic surveys of the tree of life can address important biological questions. I enumerate the significant benefits that would accrue from sequencing a broader range of taxa, as well as discuss the technical advances in sequencing and assembly methods that would allow for wide-ranging application of whole-genome analysis. Finally, I suggest that in addition to ‘big science’ survey initiatives to sequence the tree of life, a modified infrastructure-funding paradigm would better support reference genome sequence generation for research communities most in need. Copyright © 2015 Elsevier Ltd. All rights reserved.

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July 7, 2019

Comparative Analysis of the Shared Sex-Determination Region (SDR) among Salmonid Fishes.

Salmonids present an excellent model for studying evolution of young sex-chromosomes. Within the genus, Oncorhynchus, at least six independent sex-chromosome pairs have evolved, many unique to individual species. This variation results from the movement of the sex-determining gene, sdY, throughout the salmonid genome. While sdY is known to define sexual differentiation in salmonids, the mechanism of its movement throughout the genome has remained elusive due to high frequencies of repetitive elements, rDNA sequences, and transposons surrounding the sex-determining regions (SDR). Despite these difficulties, bacterial artificial chromosome (BAC) library clones from both rainbow trout and Atlantic salmon containing the sdY region have been reported. Here, we report the sequences for these BACs as well as the extended sequence for the known SDR in Chinook gained through genome walking methods. Comparative analysis allowed us to study the overlapping SDRs from three unique salmonid Y chromosomes to define the specific content, size, and variation present between the species. We found approximately 4.1 kb of orthologous sequence common to all three species, which contains the genetic content necessary for masculinization. The regions contain transposable elements that may be responsible for the translocations of the SDR throughout salmonid genomes and we examine potential mechanistic roles of each one. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Tandem repeats in rodents genome and their mapping.

Tandemly-repeated sequences represent a unique class of eukaryotic DNA. Their content in the genome of higher eukaryotes mounts to tens of percents. However, the evolution of this class of sequences is poorly-studied. In our paper, 62 families of Mus musculus tandem repeats are analyzed by bioinformatic methods, and 7 of them are analyzed by fluorescence in situ hybridization. It is shown that the same tandem repeat sets co-occure only in closely related species of mice. But even in such species we observe differences in localization on the chromosomes and the number of individual tandem repeats. With increasing evolutionary distance only some of the tandem repeat families remain common for different species. It is shown, that the use of a combination of bioinformatics and molecular biology techniques is very perspective for further studies of the evolution of tandem repeats.

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July 7, 2019

GMcloser: closing gaps in assemblies accurately with a likelihood-based selection of contig or long-read alignments.

Genome assemblies generated with next-generation sequencing (NGS) reads usually contain a number of gaps. Several tools have recently been developed to close the gaps in these assemblies with NGS reads. Although these gap-closing tools efficiently close the gaps, they entail a high rate of misassembly at gap-closing sites.We have found that the assembly error rates caused by these tools are 20-500-fold higher than the rate of errors introduced into contigs by de novo assemblers. We here describe GMcloser, a tool that accurately closes these gaps with a preassembled contig set or a long read set (i.e. error-corrected PacBio reads). GMcloser uses likelihood-based classifiers calculated from the alignment statistics between scaffolds, contigs and paired-end reads to correctly assign contigs or long reads to gap regions of scaffolds, thereby achieving accurate and efficient gap closure. We demonstrate with sequencing data from various organisms that the gap-closing accuracy of GMcloser is 3-100-fold higher than those of other available tools, with similar efficiency.GMcloser and an accompanying tool (GMvalue) for evaluating the assembly and correcting misassemblies except SNPs and short indels in the assembly are available at https://sourceforge.net/projects/gmcloser/.shunichi.kosugi@riken.jpSupplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization.

The evolutionary origins of lingulid brachiopods and their calcium phosphate shells have been obscure. Here we decode the 425-Mb genome of Lingula anatina to gain insights into brachiopod evolution. Comprehensive phylogenomic analyses place Lingula close to molluscs, but distant from annelids. The Lingula gene number has increased to ~34,000 by extensive expansion of gene families. Although Lingula and vertebrates have superficially similar hard tissue components, our genomic, transcriptomic and proteomic analyses show that Lingula lacks genes involved in bone formation, indicating an independent origin of their phosphate biominerals. Several genes involved in Lingula shell formation are shared by molluscs. However, Lingula has independently undergone domain combinations to produce shell matrix collagens with EGF domains and carries lineage-specific shell matrix proteins. Gene family expansion, domain shuffling and co-option of genes appear to be the genomic background of Lingula’s unique biomineralization. This Lingula genome provides resources for further studies of lophotrochozoan evolution.

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July 7, 2019

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano.

The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell-fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ~75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50 = 222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.

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July 7, 2019

CHOgenome.org 2.0: Genome resources and website updates.

Chinese hamster ovary (CHO) cells are a major host cell line for the production of therapeutic proteins, and CHO cell and Chinese hamster (CH) genomes have recently been sequenced using next-generation sequencing methods. CHOgenome.org was launched in 2011 (version 1.0) to serve as a database repository and to provide bioinformatics tools for the CHO community. CHOgenome.org (version 1.0) maintained GenBank CHO-K1 genome data, identified CHO-omics literature, and provided a CHO-specific BLAST service. Recent major updates to CHOgenome.org (version 2.0) include new sequence and annotation databases for both CHO and CH genomes, a more user-friendly website, and new research tools, including a proteome browser and a genome viewer. CHO cell-line specific sequences and annotations facilitate cell line development opportunities, several of which are discussed. Moving forward, CHOgenome.org will host the increasing amount of CHO-omics data and continue to make useful bioinformatics tools available to the CHO community. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 7, 2019

Jitterbug: somatic and germline transposon insertion detection at single-nucleotide resolution.

Transposable elements are major players in genome evolution. Transposon insertion polymorphisms can translate into phenotypic differences in plants and animals and are linked to different diseases including human cancer, making their characterization highly relevant to the study of genome evolution and genetic diseases. Here we present Jitterbug, a novel tool that identifies transposable element insertion sites at single-nucleotide resolution based on the pairedend mapping and clipped-read signatures produced by NGS alignments. Jitterbug can be easily integrated into existing NGS analysis pipelines, using the standard BAM format produced by frequently applied alignment tools (e.g. bwa, bowtie2), with no need to realign reads to a set of consensus transposon sequences. Jitterbug is highly sensitive and able to recall transposon insertions with a very high specificity, as demonstrated by benchmarks in the human and Arabidopsis genomes, and validation using long PacBio reads. In addition, Jitterbug estimates the zygosity of transposon insertions with high accuracy and can also identify somatic insertions. We demonstrate that Jitterbug can identify mosaic somatic transposon movement using sequenced tumor-normal sample pairs and allows for estimating the cancer cell fraction of clones containing a somatic TE insertion. We suggest that the independent methods we use to evaluate performance are a step towards creating a gold standard dataset for benchmarking structural variant prediction tools.

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July 7, 2019

The genome and methylome of a beetle with complex social behavior, Nicrophorus vespilloides (Coleoptera: Silphidae).

Testing for conserved and novel mechanisms underlying phenotypic evolution requires a diversity of genomes available for comparison spanning multiple independent lineages. For example, complex social behavior in insects has been investigated primarily with eusocial lineages, nearly all of which are Hymenoptera. If conserved genomic influences on sociality do exist, we need data from a wider range of taxa that also vary in their levels of sociality. Here, we present the assembled and annotated genome of the subsocial beetle Nicrophorus vespilloides, a species long used to investigate evolutionary questions of complex social behavior. We used this genome to address two questions. First, do aspects of life history, such as using a carcass to breed, predict overlap in gene models more strongly than phylogeny? We found that the overlap in gene models was similar between N. vespilloides and all other insect groups regardless of life history. Second, like other insects with highly developed social behavior but unlike other beetles, does N. vespilloides have DNA methylation? We found strong evidence for an active DNA methylation system. The distribution of methylation was similar to other insects with exons having the most methylated CpGs. Methylation status appears highly conserved; 85% of the methylated genes in N. vespilloides are also methylated in the hymentopteran Nasonia vitripennis. The addition of this genome adds a coleopteran resource to answer questions about the evolution and mechanistic basis of sociality and to address questions about the potential role of methylation in social behavior. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Unique transposon landscapes are pervasive across Drosophila melanogaster genomes.

To understand how transposon landscapes (TLs) vary across animal genomes, we describe a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) and a database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). Our analysis reveals pervasive TL diversity across cell lines and fly strains, even for identically named sub-strains from different laboratories such as the ISO1 strain used for the reference genome sequence. On average, >500 novel insertions exist in every lab strain, inbred strains of the Drosophila Genetic Reference Panel (DGRP), and fly isolates in the Drosophila Genome Nexus (DGN). A minority (<25%) of transposon families comprise the majority (>70%) of TL diversity across fly strains. A sharp contrast between insertion and depletion patterns indicates that many transposons are unique to the ISO1 reference genome sequence. Although TL diversity from fly strains reaches asymptotic limits with increasing sequencing depth, rampant TL diversity causes unsaturated detection of TLs in pools of flies. Finally, we show novel transposon insertions negatively correlate with Piwi-interacting RNA (piRNA) levels for most transposon families, except for the highly-abundant roo retrotransposon. Our study provides a useful resource for Drosophila geneticists to understand how transposons create extensive genomic diversity in fly cell lines and strains.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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