A high-quality Ixodes scapularis genome advances tick science.

Ixodes spp. and related ticks transmit prevalent infections, although knowledge of their biology and development of anti-tick measures have been hindered by the lack of a high-quality genome. In the present study, we present the assembly of a 2.23-Gb Ixodes scapularis genome by sequencing two haplotypes within one individual, complemented by chromosome-level scaffolding and full-length RNA isoform sequencing, yielding a fully reannotated genome featuring thousands of new protein-coding genes and various RNA species. Analyses of the repetitive DNA identified transposable elements, whereas the examination of tick-associated bacterial sequences yielded an improved Rickettsia buchneri genome. We demonstrate how the Ixodes genome advances tick science by contributing to new annotations, gene models and epigenetic functions, expansion of gene families, development of in-depth proteome catalogs and deciphering of genetic variations in wild ticks. Overall, we report critical genetic resources and biological insights impacting our understanding of tick biology and future interventions against tick-transmitted infections.

Genomic Resources to Guide Improvement of the Shea Tree.

A defining component of agroforestry parklands across Sahelo-Sudanian Africa (SSA), the shea tree (Vitellaria paradoxa) is central to sustaining local livelihoods and the farming environments of rural communities. Despite its economic and cultural value, however, not to mention the ecological roles it plays as a dominant parkland species, shea remains semi-domesticated with virtually no history of systematic genetic improvement. In truth, shea's extended juvenile period makes traditional breeding approaches untenable; but the opportunity for genome-assisted breeding is immense, provided the foundational resources are available. Here we report the development and public release of such resources. Using the FALCON-Phase workflow, 162.6 Gb of long-read PacBio sequence data were assembled into a 658.7 Mbp, chromosome-scale reference genome annotated with 38,505 coding genes. Whole genome duplication (WGD) analysis based on this gene space revealed clear signatures of two ancient WGD events in shea's evolutionary past, one prior to the Astrid-Rosid divergence (116-126 Mya) and the other at the root of the order Ericales (65-90 Mya). In a first genome-wide look at the suite of fatty acid (FA) biosynthesis genes that likely govern stearin content, the primary determinant of shea butter quality, relatively high copy numbers of six key enzymes were found (KASI, KASIII, FATB, FAD2, FAD3, and FAX2), some likely originating in shea's more recent WGD event. To help translate these findings into practical tools for characterization, selection, and genome-wide association studies (GWAS), resequencing data from a shea diversity panel was used to develop a database of more than 3.5 million functionally annotated, physically anchored SNPs. Two smaller, more curated sets of suggested SNPs, one for GWAS (104,211 SNPs) and the other targeting FA biosynthesis genes (90 SNPs), are also presented. With these resources, the hope is to support national programs across the shea belt in the strategic, genome-enabled conservation and long-term improvement of the shea tree for SSA.

Extended haplotype-phasing of long-read de novo genome assemblies using Hi-C.

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. To date, these assemblies have been best created with complex protocols, such as cultured cells that contain a single-haplotype (haploid) genome, single cells where haplotypes are separated, or co-sequencing of parental genomes in a trio-based approach. These approaches are impractical in most situations. To address this issue, we present FALCON-Phase, a phasing tool that uses ultra-long-range Hi-C chromatin interaction data to extend phase blocks of partially-phased diploid assembles to chromosome or scaffold scale. FALCON-Phase uses the inherent phasing information in Hi-C reads, skipping variant calling, and reduces the computational complexity of phasing. Our method is validated on three benchmark datasets generated as part of the Vertebrate Genomes Project (VGP), including human, cow, and zebra finch, for which high-quality, fully haplotype-resolved assemblies are available using the trio-based approach. FALCON-Phase is accurate without having parental data and performance is better in samples with higher heterozygosity. For cow and zebra finch the accuracy is 97% compared to 80-91% for human. FALCON-Phase is applicable to any draft assembly that contains long primary contigs and phased associate contigs.

A draft phased assembly of the diploid Cascade hop (Humulus lupulus) genome.

Hop (Humulus lupulus L. var Lupulus) is a diploid, dioecious plant with a history of cultivation spanning more than one thousand years. Hop cones are valued for their use in brewing and contain compounds of therapeutic interest including xanthohumol. Efforts to determine how biochemical pathways responsible for desirable traits are regulated have been challenged by the large (2.8 Gb), repetitive, and heterozygous genome of hop. We present a draft haplotype-phased assembly of the Cascade cultivar genome. Our draft assembly and annotation of the Cascade genome is the most extensive representation of the hop genome to date. PacBio long-read sequences from hop were assembled with FALCON and partially phased with FALCON-Unzip. Comparative analysis of haplotype sequences provides insight into selective pressures that have driven evolution in hop. We discovered genes with greater sequence divergence enriched for stress-response, growth, and flowering functions in the draft phased assembly. With improved resolution of long terminal retrotransposons (LTRs) due to long-read sequencing, we found that hop is over 70% repetitive. We identified a homolog of cannabidiolic acid synthase (CBDAS) that is expressed in multiple tissues. The approaches we developed to analyze the draft phased assembly serve to deepen our understanding of the genomic landscape of hop and may have broader applicability to the study of other large, complex genomes.

Haplotype-resolved genomes provide insights into structural variation and gene content in Angus and Brahman cattle.

Inbred animals were historically chosen for genome analysis to circumvent assembly issues caused by haplotype variation but this resulted in a composite of the two genomes. Here we report a haplotype-aware scaffolding and polishing pipeline which was used to create haplotype-resolved, chromosome-level genome assemblies of Angus (taurine) and Brahman (indicine) cattle subspecies from contigs generated by the trio binning method. These assemblies reveal structural and copy number variants that differentiate the subspecies and that variant detection is sensitive to the specific reference genome chosen. Six genes with immune related functions have additional copies in the indicine compared with taurine lineage and an indicus-specific extra copy of fatty acid desaturase is under positive selection. The haplotyped genomes also enable transcripts to be phased to detect allele-specific expression. This work exemplifies the value of haplotype-resolved genomes to better explore evolutionary and functional variations.

A high-quality genome assembly from a single, field-collected spotted lanternfly (Lycorma delicatula) using the PacBio Sequel II system.

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.

A High-Quality De novo Genome Assembly from a Single Mosquito Using PacBio Sequencing.

A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

Chromosome-level assembly of the water buffalo genome surpasses human and goat genomes in sequence contiguity.

Rapid innovation in sequencing technologies and improvement in assembly algorithms have enabled the creation of highly contiguous mammalian genomes. Here we report a chromosome-level assembly of the water buffalo (Bubalus bubalis) genome using single-molecule sequencing and chromatin conformation capture data. PacBio Sequel reads, with a mean length of 11.5 kb, helped to resolve repetitive elements and generate sequence contiguity. All five B. bubalis sub-metacentric chromosomes were correctly scaffolded with centromeres spanned. Although the index animal was partly inbred, 58% of the genome was haplotype-phased by FALCON-Unzip. This new reference genome improves the contig N50 of the previous short-read based buffalo assembly more than a thousand-fold and contains only 383 gaps. It surpasses the human and goat references in sequence contiguity and facilitates the annotation of hard to assemble gene clusters such as the major histocompatibility complex (MHC).

Gene flow mediates the role of sex chromosome meiotic drive during complex speciation.

During speciation, sex chromosomes often accumulate interspecific genetic incompatibilities faster than the rest of the genome. The drive theory posits that sex chromosomes are susceptible to recurrent bouts of meiotic drive and suppression, causing the evolutionary build-up of divergent cryptic sex-linked drive systems and, incidentally, genetic incompatibilities. To assess the role of drive during speciation, we combine high-resolution genetic mapping of X-linked hybrid male sterility with population genomics analyses of divergence and recent gene flow between the fruitfly species, Drosophila mauritiana and D. simulans. Our findings reveal a high density of genetic incompatibilities and a corresponding dearth of gene flow on the X chromosome. Surprisingly, we find that a known drive element recently migrated between species and, rather than contributing to interspecific divergence, caused a strong reduction in local sequence divergence, undermining the evolution of hybrid sterility. Gene flow can therefore mediate the effects of selfish genetic elements during speciation.

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.

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.

Molecular Evolution at a Meiosis Gene Mediates Species Differences in the Rate and Patterning of Recombination.

Crossing over between homologous chromosomes during meiosis repairs programmed DNA double-strand breaks, ensures proper segregation at meiosis I [1], shapes the genomic distribution of nucleotide variability in populations, and enhances the efficacy of natural selection among genetically linked sites [2]. Between closely related Drosophila species, large differences exist in the rate and chromosomal distribution of crossing over. Little, however, is known about the molecular genetic changes or population genetic forces that mediate evolved differences in recombination between species [3, 4]. Here, we show that a meiosis gene with a history of rapid evolution acts as a trans-acting modifier of species differences in crossing over. In transgenic flies, the dicistronic gene, mei-217/mei-218, recapitulates a large part of the species differences in the rate and chromosomal distribution of crossing over. These phenotypic differences appear to result from changes in protein sequence not gene expression. Our population genetics analyses show that the protein-coding sequence of mei-218, but not mei-217, has a history of recurrent positive natural selection. By modulating the intensity of centromeric and telomeric suppression of crossing over, evolution at mei-217/-218 has incidentally shaped gross differences in the chromosomal distribution of nucleotide variability between species. We speculate that recurrent bouts of adaptive evolution at mei-217/-218 might reflect a history of coevolution with selfish genetic elements.

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.

A new method to scan genomes for introgression in a secondary contact model.

Secondary contact between divergent populations or incipient species may result in the exchange and introgression of genomic material. We develop a simple DNA sequence measure, called Gmin, which is designed to identify genomic regions experiencing introgression in a secondary contact model. Gmin is defined as the ratio of the minimum between-population number of nucleotide differences in a genomic window to the average number of between-population differences. Although it is conceptually simple, one advantage of Gmin is that it is computationally inexpensive relative to model-based methods for detecting gene flow and it scales easily to the level of whole-genome analysis. We compare the sensitivity and specificity of Gmin to those of the widely used index of population differentiation, FST, and suggest a simple statistical test for identifying genomic outliers. Extensive computer simulations demonstrate that Gmin has both greater sensitivity and specificity for detecting recent introgression than does FST. Furthermore, we find that the sensitivity of Gmin is robust with respect to both the population mutation and recombination rates. Finally, a scan of Gmin across the X chromosome of Drosophila melanogaster identifies candidate regions of introgression between sub-Saharan African and cosmopolitan populations that were previously missed by other methods. These results show that Gmin is a biologically straightforward, yet powerful, alternative to FST, as well as to more computationally intensive model-based methods for detecting gene flow.

Genome diversity and divergence in Drosophila mauritiana: multiple signatures of faster X evolution.

Drosophila mauritiana is an Indian Ocean island endemic species that diverged from its two sister species, Drosophila simulans and Drosophila sechellia, approximately 240,000 years ago. Multiple forms of incomplete reproductive isolation have evolved among these species, including sexual, gametic, ecological, and intrinsic postzygotic barriers, with crosses among all three species conforming to Haldane's rule: F(1) hybrid males are sterile and F(1) hybrid females are fertile. Extensive genetic resources and the fertility of hybrid females have made D. mauritiana, in particular, an important model for speciation genetics. Analyses between D. mauritiana and both of its siblings have shown that the X chromosome makes a disproportionate contribution to hybrid male sterility. But why the X plays a special role in the evolution of hybrid sterility in these, and other, species remains an unsolved problem. To complement functional genetic analyses, we have investigated the population genomics of D. mauritiana, giving special attention to differences between the X and the autosomes. We present a de novo genome assembly of D. mauritiana annotated with RNAseq data and a whole-genome analysis of polymorphism and divergence from ten individuals. Our analyses show that, relative to the autosomes, the X chromosome has reduced nucleotide diversity but elevated nucleotide divergence; an excess of recurrent adaptive evolution at its protein-coding genes; an excess of recent, strong selective sweeps; and a large excess of satellite DNA. Interestingly, one of two centimorgan-scale selective sweeps on the D. mauritiana X chromosome spans a region containing two sex-ratio meiotic drive elements and a high concentration of satellite DNA. Furthermore, genes with roles in reproduction and chromosome biology are enriched among genes that have histories of recurrent adaptive protein evolution. Together, these genome-wide analyses suggest that genetic conflict and frequent positive natural selection on the X chromosome have shaped the molecular evolutionary history of D. mauritiana, refining our understanding of the possible causes of the large X-effect in speciation.

A selective sweep across species boundaries in Drosophila.

Adaptive mutations that accumulate during species divergence are likely to contribute to reproductive incompatibilities and hinder gene flow; however, there may also be a class of mutations that are generally advantageous and can spread across species boundaries. In this study, we characterize a 15 kb region on chromosome 3R that has introgressed from the cosmopolitan generalist species Drosophila simulans into the island endemic D. sechellia, which is an ecological specialist. The introgressed haplotype is fixed in D. sechellia over almost the entirety of the resequenced region, whereas a core region of the introgressed haplotype occurs at high frequency in D. simulans. The observed patterns of nucleotide variation and linkage disequilibrium are consistent with a recently completed selective sweep in D. sechellia and an incomplete sweep in D. simulans. Independent estimates of both the time to the introgression and sweep events are all close to 10,000 years before the present. Interestingly, the most likely target of selection is a highly occupied transcription factor binding region. This work confirms that it is possible for mutations to be globally advantageous, despite their occurrence in divergent genomic and ecological backgrounds.

Genome sequencing reveals complex speciation in the Drosophila simulans clade.

The three species of the Drosophila simulans clade--the cosmopolitan species, D. simulans, and the two island endemic species, D. mauritiana and D. sechellia--are important models in speciation genetics, but some details of their phylogenetic and speciation history remain unresolved. The order and timing of speciation are disputed, and the existence, magnitude, and timing of gene flow among the three species remain unclear. Here we report on the analysis of a whole-genome four-species sequence alignment that includes all three D. simulans clade species as well as the D. melanogaster reference sequence. The alignment comprises novel, paired short-read sequence data from a single highly inbred line each from D. simulans, D. mauritiana, and D. sechellia. We are unable to reject a species phylogeny with a basal polytomy; the estimated age of the polytomy is 242,000 yr before the present. However, we also find that up to 4.6% of autosomal and 2.2% of X-linked regions have evolutionary histories consistent with recent gene flow between the mainland species (D. simulans) and the two island endemic species (D. mauritiana and D. sechellia). Our findings thus show that gene flow has occurred throughout the genomes of the D. simulans clade species despite considerable geographic, ecological, and intrinsic reproductive isolation. Last, our analysis of lineage-specific changes confirms that the D. sechellia genome has experienced a significant excess of slightly deleterious changes and a dearth of presumed favorable changes. The relatively reduced efficacy of natural selection in D. sechellia is consistent with its derived, persistently reduced historical effective population size.

Sex chromosome-specific regulation in the Drosophila male germline but little evidence for chromosomal dosage compensation or meiotic inactivation.

The evolution of heteromorphic sex chromosomes (e.g., XY in males or ZW in females) has repeatedly elicited the evolution of two kinds of chromosome-specific regulation: dosage compensation--the equalization of X chromosome gene expression in males and females--and meiotic sex chromosome inactivation (MSCI)--the transcriptional silencing and heterochromatinization of the X during meiosis in the male (or Z in the female) germline. How the X chromosome is regulated in the Drosophila melanogaster male germline is unclear. Here we report three new findings concerning gene expression from the X in Drosophila testes. First, X chromosome-wide dosage compensation appears to be absent from most of the Drosophila male germline. Second, microarray analysis provides no evidence for X chromosome-specific inactivation during meiosis. Third, we confirm the previous discovery that the expression of transgene reporters driven by autosomal spermatogenesis-specific promoters is strongly reduced when inserted on the X chromosome versus the autosomes; but we show that this chromosomal difference in expression is established in premeiotic cells and persists in meiotic cells. The magnitude of the X-autosome difference in transgene expression cannot be explained by the absence of dosage compensation, suggesting that a previously unrecognized mechanism limits expression from the X during spermatogenesis in Drosophila. These findings help to resolve several previously conflicting reports and have implications for patterns of genome evolution and speciation in Drosophila.

Recurrent selection on the Winters sex-ratio genes in Drosophila simulans.

Selfish genes, such as meiotic drive elements, propagate themselves through a population without increasing the fitness of host organisms. X-linked (or Y-linked) meiotic drive elements reduce the transmission of the Y (X) chromosome and skew progeny and population sex ratios, leading to intense conflict among genomic compartments. Drosophila simulans is unusual in having a least three distinct systems of X chromosome meiotic drive. Here, we characterize naturally occurring genetic variation at the Winters sex-ratio driver (Distorter on the X or Dox), its progenitor gene (Mother of Dox or MDox), and its suppressor gene (Not Much Yang or Nmy), which have been previously mapped and characterized. We survey three North American populations as well as 13 globally distributed strains and present molecular polymorphism data at the three loci. We find that all three genes show signatures of selection in North America, judging from levels of polymorphism and skews in the site-frequency spectrum. These signatures likely result from the biased transmission of the driver and selection on the suppressor for the maintenance of equal sex ratios. Coalescent modeling indicates that the timing of selection is more recent than the age of the alleles, suggesting that the driver and suppressor are coevolving under an evolutionary "arms race." None of the Winters sex-ratio genes are fixed in D. simulans, and at all loci we find ancestral alleles, which lack the gene insertions and exhibit high levels of nucleotide polymorphism compared to the derived alleles. In addition, we find several "null" alleles that have mutations on the derived Dox background, which result in loss of drive function. We discuss the possible causes of the maintenance of presence-absence polymorphism in the Winters sex-ratio genes.