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July 19, 2019

Winding paths to simplicity: genome evolution in facultative insect symbionts.

Symbiosis between organisms is an important driving force in evolution. Among the diverse relationships described, extensive progress has been made in insect-bacteria symbiosis, which improved our understanding of the genome evolution in host-associated bacteria. Particularly, investigations on several obligate mutualists have pushed the limits of what we know about the minimal genomes for sustaining cellular life. To bridge the gap between those obligate symbionts with extremely reduced genomes and their non-host-restricted ancestors, this review focuses on the recent progress in genome characterization of facultative insect symbionts. Notable cases representing various types and stages of host associations, including those from multiple genera in the family Enterobacteriaceae (class Gammaproteobacteria), Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), are discussed. Although several general patterns of genome reduction associated with the adoption of symbiotic relationships could be identified, extensive variation was found among these facultative symbionts. These findings are incorporated into the established conceptual frameworks to develop a more detailed evolutionary model for the discussion of possible trajectories. In summary, transitions from facultative to obligate symbiosis do not appear to be a universal one-way street; switches between hosts and lifestyles (e.g. commensalism, parasitism or mutualism) occur frequently and could be facilitated by horizontal gene transfer. © FEMS 2016.

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July 19, 2019

Ribbon: Visualizing complex genome alignments and structural variation

Visualization has played an extremely important role in the current genomic revolution to inspect and understand variants, expression patterns, evolutionary changes, and a number of other relationships. However, most of the information in read-to-reference or genome-genome alignments is lost for structural variations in the one-dimensional views of most genome browsers showing only reference coordinates. Instead, structural variations captured by long reads or assembled contigs often need more context to understand, including alignments and other genomic information from multiple chromosomes. We have addressed this problem by creating Ribbon (genomeribbon.com) an interactive online visualization tool that displays alignments along both reference and query sequences, along with any associated variant calls in the sample. This way Ribbon shows patterns in alignments of many reads across multiple chromosomes, while allowing detailed inspection of individual reads (Supplementary Note 1). For example, here we show a gene fusion in the SK-BR-3 breast cancer cell line linking the genes CYTH1 and EIF3H. While it has been found in the transcriptome previously, genome sequencing did not identify a direct chromosomal fusion between these two genes. After SMRT sequencing, Ribbon shows that there are indeed long reads that span from one gene to the other, going through not one but two variants, for the first time showing the genomic link between these two genes (Figure 1a). More gene fusions of this cancer cell line are investigated in Supplementary Note 2. Figure 1b shows another complex event in this sample made simple in Ribbon: the translocation of a 4.4 kb sequence deleted from chr19 and inserted into chr16 (Figure 1b). Thus, Ribbon enables understanding of complex variants, and it may also help in the detection of sequencing and sample preparation issues, testing of aligners and variant-callers, and rapid curation of structural variant candidates (Supplementary Note 3). In addition to SAM and BAM files with long, short, or paired-end reads, Ribbon can also load coordinate files from whole genome aligners such as MUMmer. Therefore, Ribbon can be used to test assembly algorithms or inspect the similarity between species. Supplementary Note 4 shows a comparison of gorilla and human genomes using Ribbon, highlighting major structural differences. In conclusion, Ribbon is a powerful interactive web tool for viewing complex genomic alignments.

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July 19, 2019

The deep origin and recent loss of venom toxin genes in rattlesnakes.

The genetic origin of novel traits is a central but challenging puzzle in evolutionary biology. Among snakes, phospholipase A2 (PLA2)-related toxins have evolved in different lineages to function as potent neurotoxins, myotoxins, or hemotoxins. Here, we traced the genomic origin and evolution of PLA2 toxins by examining PLA2 gene number, organization, and expression in both neurotoxic and non-neurotoxic rattlesnakes. We found that even though most North American rattlesnakes do not produce neurotoxins, the genes of a specialized heterodimeric neurotoxin predate the origin of rattlesnakes and were present in their last common ancestor (~22 mya). The neurotoxin genes were then deleted independently in the lineages leading to the Western Diamondback (Crotalus atrox) and Eastern Diamondback (C. adamanteus) rattlesnakes (~6 mya), while a PLA2 myotoxin gene retained in C. atrox was deleted from the neurotoxic Mojave rattlesnake (C. scutulatus; ~4 mya). The rapid evolution of PLA2 gene number appears to be due to transposon invasion that provided a template for non-allelic homologous recombination. Copyright © 2016 Elsevier Ltd. All rights reserved.

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July 19, 2019

Rapid functional and sequence differentiation of a tandemly repeated species-specific multigene family in Drosophila.

Gene clusters of recently duplicated genes are hotbeds for evolutionary change. However, our understanding of how mutational mechanisms and evolutionary forces shape the structural and functional evolution of these clusters is hindered by the high sequence identity among the copies, which typically results in their inaccurate representation in genome assemblies. The presumed testis-specific, chimeric gene Sdic originated, and tandemly expanded in Drosophila melanogaster, contributing to increased male-male competition. Using various types of massively parallel sequencing data, we studied the organization, sequence evolution, and functional attributes of the different Sdic copies. By leveraging long-read sequencing data, we uncovered both copy number and order differences from the currently accepted annotation for the Sdic region. Despite evidence for pervasive gene conversion affecting the Sdic copies, we also detected signatures of two episodes of diversifying selection, which have contributed to the evolution of a variety of C-termini and miRNA binding site compositions. Expression analyses involving RNA-seq datasets from 59 different biological conditions revealed distinctive expression breadths among the copies, with three copies being transcribed in females, opening the possibility to a sexually antagonistic effect. Phenotypic assays using Sdic knock-out strains indicated that should this antagonistic effect exist, it does not compromise female fertility. Our results strongly suggest that the genome consolidation of the Sdic gene cluster is more the result of a quick exploration of different paths of molecular tinkering by different copies than a mere dosage increase, which could be a recurrent evolutionary outcome in the presence of persistent sexual selection. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 19, 2019

Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

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July 19, 2019

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome.

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ~400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.

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July 19, 2019

Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation.

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either PacBio or Oxford Nanopore technologies, and achieves a contig NG50 of greater than 21 Mbp on both human and Drosophila melanogaster PacBio datasets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes. Published by Cold Spring Harbor Laboratory Press.

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July 19, 2019

A golden goat genome

The newly described de novo goat genome sequence is the most contiguous diploid vertebrate assembly generated thus far using whole-genome assembly and scaffolding methods. The contiguity of this assembly is approaching that of the finished human and mouse genomes and suggests an affordable roadmap to high-quality references for thousands of species.

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July 19, 2019

Single-molecule sequencing resolves the detailed structure of complex satellite DNA loci in Drosophila melanogaster.

Highly repetitive satellite DNA (satDNA) repeats are found in most eukaryotic genomes. SatDNAs are rapidly evolving and have roles in genome stability and chromosome segregation. Their repetitive nature poses a challenge for genome assembly and makes progress on the detailed study of satDNA structure difficult. Here, we use single-molecule sequencing long reads from Pacific Biosciences (PacBio) to determine the detailed structure of all major autosomal complex satDNA loci in Drosophila melanogaster, with a particular focus on the 260-bp and Responder satellites. We determine the optimal de novo assembly methods and parameter combinations required to produce a high-quality assembly of these previously unassembled satDNA loci and validate this assembly using molecular and computational approaches. We determined that the computationally intensive PBcR-BLASR assembly pipeline yielded better assemblies than the faster and more efficient pipelines based on the MHAP hashing algorithm, and it is essential to validate assemblies of repetitive loci. The assemblies reveal that satDNA repeats are organized into large arrays interrupted by transposable elements. The repeats in the center of the array tend to be homogenized in sequence, suggesting that gene conversion and unequal crossovers lead to repeat homogenization through concerted evolution, although the degree of unequal crossing over may differ among complex satellite loci. We find evidence for higher-order structure within satDNA arrays that suggest recent structural rearrangements. These assemblies provide a platform for the evolutionary and functional genomics of satDNAs in pericentric heterochromatin. © 2017 Khost et al.; Published by Cold Spring Harbor Laboratory Press.

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July 19, 2019

A new chicken genome assembly provides insight into avian genome structure.

The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3), built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb, including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31, and 33, and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 protein-coding) over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B, encompassing two segments: one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts. Copyright © 2017 Warren et al.

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July 19, 2019

Reduction in chromosome mobility accompanies nuclear organization during early embryogenesis in Caenorhabditis elegans.

In differentiated cells, chromosomes are packed inside the cell nucleus in an organised fashion. In contrast, little is known about how chromosomes are packed in undifferentiated cells and how nuclear organization changes during development. To assess changes in nuclear organization during the earliest stages of development, we quantified the mobility of a pair of homologous chromosomal loci in the interphase nuclei of Caenorhabditis elegans embryos. The distribution of distances between homologous loci was consistent with a random distribution up to the 8-cell stage but not at later stages. The mobility of the loci was significantly reduced from the 2-cell to the 48-cell stage. Nuclear foci corresponding to epigenetic marks as well as heterochromatin and the nucleolus also appeared around the 8-cell stage. We propose that the earliest global transformation in nuclear organization occurs at the 8-cell stage during C. elegans embryogenesis.

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July 19, 2019

New technologies boost genome quality.

Three years ago, Erich Jarvis helped mastermind a massive DNA sequenc- ing effort that netted genomes for more than 40 bird species and produced a better avian family tree. But when he tried to compare the avian genomes to those of other species to learn about the evolution and function of several key brain genes, he was stymied. His team found that gene sequences from most of the comparison species—even humans—were incomplete, missing, or misplaced in the larger genome. The group had to resequence sections of sev- eral genomes to get the needed data, delaying their project many months.

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July 19, 2019

Omics approaches to study gene regulatory networks for development in echinoderms.

Gene regulatory networks (GRNs) describe the interactions for a developmental process at a given time and space. Historically, perturbation experiments represent one of the key methods for analyzing and reconstructing a GRN, and the GRN governing early development in the sea urchin embryo stands as one of the more deeply dissected so far. As technology progresses, so do the methods used to address different biological questions. Next-generation sequencing (NGS) has become a standard experimental technique for genome and transcriptome sequencing and studies of protein-DNA interactions and DNA accessibility. While several efforts have been made toward the integration of different omics approaches for the study of the regulatory genome in many animals, in a few cases, these are applied with the purpose of reconstructing and experimentally testing developmental GRNs. Here, we review emerging approaches integrating multiple NGS technologies for the prediction and validation of gene interactions within echinoderm GRNs. These approaches can be applied to both ‘model’ and ‘non-model’ organisms. Although a number of issues still need to be addressed, advances in NGS applications, such as assay for transposase-accessible chromatin sequencing, combined with the availability of embryos belonging to different species, all separated by various evolutionary distances and accessible to experimental regulatory biology, place echinoderms in an unprecedented position for the reconstruction and evolutionary comparison of developmental GRNs. We conclude that sequencing technologies and integrated omics approaches allow the examination of GRNs on a genome-wide scale only if biological perturbation and cis-regulatory analyses are experimentally accessible, as in the case of echinoderm embryos.© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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July 19, 2019

Characterisation of MHC class I genes in the koala.

Koala (Phascolarctos cinereus) populations are on the decline across the majority of Australia’s mainland. Two major diseases threatening the long-term survival of affected koala populations are caused by obligate intracellular pathogens: Chlamydia and koala retrovirus (KoRV). To improve our understanding of the koala immune system, we characterised their major histocompatibility complex (MHC) class I genes, which are centrally involved in presenting foreign peptides derived from intracellular pathogens to cytotoxic T cells. A total of 11 class I genes were identified in the koala genome. Three genes, Phci-UA, UB and UC, showed relatively high genetic variability and were expressed in all 12 examined tissues, whereas the other eight genes had tissue-specific expression and limited polymorphism. Evidence of diversifying selection was detected in Phci-UA and UC, while gene conversion may have played a role in creating new alleles at Phci-UB. We propose that Phci-UA, UB and UC are likely classical MHC genes of koalas, and further research is needed to understand their role in koala chlamydial and KoRV infections.

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July 19, 2019

De novo PacBio long-read and phased avian genome assemblies correct and add to reference genes generated with intermediate and short reads.

Reference-quality genomes are expected to provide a resource for studying gene structure, function, and evolution. However, often genes of interest are not completely or accurately assembled, leading to unknown errors in analyses or additional cloning efforts for the correct sequences. A promising solution is long-read sequencing. Here we tested PacBio-based long-read sequencing and diploid assembly for potential improvements to the Sanger-based intermediate-read zebra finch reference and Illumina-based short-read Anna’s hummingbird reference, 2 vocal learning avian species widely studied in neuroscience and genomics. With DNA of the same individuals used to generate the reference genomes, we generated diploid assemblies with the FALCON-Unzip assembler, resulting in contigs with no gaps in the megabase range, representing 150-fold and 200-fold improvements over the current zebra finch and hummingbird references, respectively. These long-read and phased assemblies corrected and resolved what we discovered to be numerous misassemblies in the references, including missing sequences in gaps, erroneous sequences flanking gaps, base call errors in difficult-to-sequence regions, complex repeat structure errors, and allelic differences between the 2 haplotypes. These improvements were validated by single long-genome and transcriptome reads and resulted for the first time in completely resolved protein-coding genes widely studied in neuroscience and specialized in vocal learning species. These findings demonstrate the impact of long reads, sequencing of previously difficult-to-sequence regions, and phasing of haplotypes on generating the high-quality assemblies necessary for understanding gene structure, function, and evolution.© The Authors 2017. Published by Oxford University Press.

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