Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm.

Haplotype-resolved de novo assembly is the ultimate solution to the study of sequence variations in a genome. However, existing algorithms either collapse heterozygous alleles into one consensus copy or fail to cleanly separate the haplotypes to produce high-quality phased assemblies. Here we describe hifiasm, a de novo assembler that takes advantage of long high-fidelity sequence reads to faithfully represent the haplotype information in a phased assembly graph. Unlike other graph-based assemblers that only aim to maintain the contiguity of one haplotype, hifiasm strives to preserve the contiguity of all haplotypes. This feature enables the development of a graph trio binning algorithm that greatly advances over standard trio binning. On three human and five nonhuman datasets, including California redwood with a ~30-Gb hexaploid genome, we show that hifiasm frequently delivers better assemblies than existing tools and consistently outperforms others on haplotype-resolved assembly.

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads.

The sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective standalone technology for de novo assembly of human genomes.

Phased diploid genome assembly with single-molecule real-time sequencing.

While genome assembly projects have been successful in many haploid and inbred species, the assembly of noninbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short- or long-read approaches. The phased diploid assembly enabled the study of haplotype structure and heterozygosities between homologous chromosomes, including the identification of widespread heterozygous structural variation within coding sequences.

Dinoflagellate phylogeny revisited: using ribosomal proteins to resolve deep branching dinoflagellate clades.

The alveolates are composed of three major lineages, the ciliates, dinoflagellates, and apicomplexans. Together these 'protist' taxa play key roles in primary production and ecology, as well as in illness of humans and other animals. The interface between the dinoflagellate and apicomplexan clades has been an area of recent discovery, blurring the distinction between these two clades. Moreover, phylogenetic analysis has yet to determine the position of basal dinoflagellate clades hence the deepest branches of the dinoflagellate tree currently remain unresolved. Large-scale mRNA sequencing was applied to 11 species of dinoflagellates, including strains of the syndinean genera Hematodinium and Amoebophrya, parasites of crustaceans and dinoflagellates, respectively, to optimize and update the dinoflagellate tree. From the transcriptome-scale data a total of 73 ribosomal protein-coding genes were selected for phylogeny. After individual gene orthology assessment, the genes were concatenated into a >15,000 amino acid alignment with 76 taxa from dinoflagellates, apicomplexans, ciliates, and the outgroup heterokonts. Overall the tree was well resolved and supported, when the data was subsampled with gblocks or constraint trees were tested with the approximately unbiased test. The deepest branches of the dinoflagellate tree can now be resolved with strong support, and provides a clearer view of the evolution of the distinctive traits of dinoflagellates.

Assembly and annotation of 2 high-quality columbid reference genomes from sequencing of a Columba livia × Columba guinea F1 hybrid.

Pigeons and doves (family Columbidae) are one of the most diverse extant avian lineages, and many species have served as key models for evolutionary genomics, developmental biology, physiology, and behavioral studies. Building genomic resources for columbids is essential to further many of these studies. Here, we present high-quality genome assemblies and annotations for 2 columbid species, Columba livia and Columba guinea. We simultaneously assembled C. livia and C. guinea genomes from long-read sequencing of a single F1 hybrid individual. The new C. livia genome assembly (Cliv_3) shows improved completeness and contiguity relative to Cliv_2.1, with an annotation incorporating long-read IsoSeq data for more accurate gene models. Intensive selective breeding of C. livia has given rise to hundreds of breeds with diverse morphological and behavioral characteristics, and Cliv_3 offers improved tools for mapping the genomic architecture of interesting traits. The C. guinea genome assembly is the first for this species and is a new resource for avian comparative genomics. Together, these assemblies and annotations provide improved resources for functional studies of columbids and avian comparative genomics in general.

Assembly and annotation of two high-quality columbid reference genomes from sequencing of a Columba livia x Columba guinea F1 hybrid.

Pigeons and doves (family Columbidae) are one of the most diverse extant avian lineages, and many species have served as key models for evolutionary genomics, developmental biology, physiology, and behavioral studies. Building genomic resources for colubids is essential to further many of these studies. Here, we present high-quality genome assemblies and annotations for two columbid species, Columba livia and C. guinea. We simultaneously assembled C. livia and C. guinea genomes from long-read sequencing of a single F1 hybrid individual. The new C. livia genome assembly (Cliv_3) shows improved completeness and contiguity relative to Cliv_2.1, with an annotation incorporating long-read IsoSeq data for more accurate gene models. Intensive selective breeding of C. livia has given rise to hundreds of breeds with diverse morphological and behavioral characteristics, and Cliv_3 offers improved tools for mapping the genomic architecture of interesting traits. The C. guinea genome assembly is the first for this species and is a new resource for avian comparative genomics. Together, these assemblies and annotations provide improved resources for functional studies of columbids and avian comparative genomics in general.

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.

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.

Isolation by distance across the Hawaiian Archipelago in the reef-building coral Porites lobata.

There is an ongoing debate on the scale of pelagic larval dispersal in promoting connectivity among populations of shallow, benthic marine organisms. The linearly arranged Hawaiian Islands are uniquely suited to study scales of population connectivity and have been used extensively as a natural laboratory in terrestrial systems. Here, we focus on Hawaiian populations of the lobe coral Porites lobata, an ecosystem engineer of shallow reefs throughout the Pacific. Patterns of recent gene flow and population structure in P. lobata samples (n = 318) from two regions, the Hawaiian Islands (n = 10 sites) and from their nearest neighbour Johnston Atoll, were analysed with nine microsatellite loci. Despite its massive growth form, ∼ 6% of the samples from both regions were the product of asexual reproduction via fragmentation. Cluster analysis and measures of genetic differentiation indicated that P. lobata from the Hawaiian Islands are strongly isolated from those on Johnston Atoll (F(ST) = 0.311; P < 0.001), with the descendants of recent migrants (n = 6) being clearly identifiable. Within the Hawaiian Islands, P. lobata conforms to a pattern of isolation by distance. Here, over 37% (P = 0.001) of the variation in genetic distance was explained by geographical distance. This pattern indicates that while the majority of ongoing gene flow in Hawaiian P. lobata occurs among geographically proximate reefs, inter-island distances are insufficient to generate strong population structure across the archipelago.

Fully Phased Sequence of a Diploid Human Genome Determined de Novo from the DNA of a Single Individual.

In recent years, improved sequencing technology and computational tools have made de novo genome assembly more accessible. Many approaches, however, generate either an unphased or only partially resolved representation of a diploid genome, in which polymorphisms are detected but not assigned to one or the other of the homologous chromosomes. Yet chromosomal phase information is invaluable for the understanding of phenotypic trait inheritance in the cases of compound heterozygosity, allele-specific expression or cis-acting variants. Here we use a combination of tools and sequencing technologies to generate a de novo diploid assembly of the human primary cell line WI-38. First, data from PacBio single molecule sequencing and Bionano Genomics optical mapping were combined to generate an unphased assembly. Next, 10x Genomics linked reads were combined with the hybrid assembly to generate a partially phased assembly. Lastly, we developed and optimized methods to use short-read (Illumina) sequencing of flow cytometry-sorted metaphase chromosomes to provide phase information. The final genome assembly was almost fully (94%) phased with the addition of approximately 2.5-fold coverage of Illumina data from the sequenced metaphase chromosomes. The diploid nature of the final de novo genome assembly improved the resolution of structural variants between the WI-38 genome and the human reference genome. The phased WI-38 sequence data are available for browsing and download at wi38.research.calicolabs.com. Our work shows that assembling a completely phased diploid genome de novo from the DNA of a single individual is now readily achievable.

Generalist dinoflagellate endosymbionts and host genotype diversity detected from mesophotic (67-100 m depths) coral Leptoseris.

BACKGROUND: Mesophotic corals (light-dependent corals in the deepest half of the photic zone at depths of 30-150 m) provide a unique opportunity to study the limits of the interactions between corals and endosymbiotic dinoflagellates in the genus Symbiodinium. We sampled Leptoseris spp. in Hawaii via manned submersibles across a depth range of 67-100 m. Both the host and Symbiodinium communities were genotyped, using a non-coding region of the mitochondrial ND5 intron (NAD5) and the nuclear ribosomal internal transcribed spacer region 2 (ITS2), respectively. RESULTS: Coral colonies harbored endosymbiotic communities dominated by previously identified shallow water Symbiodinium ITS2 types (C1_ AF333515, C1c_ AY239364, C27_ AY239379, and C1b_ AY239363) and exhibited genetic variability at mitochondrial NAD5. CONCLUSION: This is one of the first studies to examine genetic diversity in corals and their endosymbiotic dinoflagellates sampled at the limits of the depth and light gradients for hermatypic corals. The results reveal that these corals associate with generalist endosymbiont types commonly found in shallow water corals and implies that the composition of the Symbiodinium community (based on ITS2) alone is not responsible for the dominance and broad depth distribution of Leptoseris spp. The level of genetic diversity detected in the coral NAD5 suggests that there is undescribed taxonomic diversity in the genus Leptoseris from Hawaii.

Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome.

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read sequencing to detect small variants and structural variants. We estimate that 2,434 discordances are correctable mistakes in the 'genome in a bottle' (GIAB) benchmark set. Nearly all (99.64%) variants can be phased into haplotypes, further improving variant detection. De novo genome assembly using CCS reads alone produced a contiguous and accurate genome with a contig N50 of >15 megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.

Dinoflagellate expressed sequence tag data indicate massive transfer of chloroplast genes to the nuclear genome.

The peridinin-pigmented plastids of dinoflagellates are very poorly understood, in part because of the paucity of molecular data available from these endosymbiotic organelles. To identify additional gene sequences that would carry information about the biology of the peridinin-type dinoflagellate plastid and its evolutionary history, an analysis was undertaken of arbitrarily selected sequences from cDNA libraries constructed from Lingulodinium polyedrum (1012 non-redundant sequences) and Amphidinium carterae (2143). Among the two libraries 118 unique plastid-associated sequences were identified, including 30 (most from A. carterae) that are encoded in the plastid genome of the red alga Porphyra. These sequences probably represent bona fide nuclear genes, and suggest that there has been massive transfer of genes from the plastid to the nuclear genome in dinoflagellates. These data support the hypothesis that the peridinin-type plastid has a minimal genome, and provide data that contradict the hypothesis that there is an unidentified canonical genome in the peridinin-type plastid. Sequences were also identified that were probably transferred directly from the nuclear genome of the red algal endosymbiont, as well as others that are distinctive to the Alveolata. A preliminary report of these data was presented at the Botany 2002 meeting in Madison, WI.

From parent to gamete: vertical transmission of Symbiodinium (Dinophyceae) ITS2 sequence assemblages in the reef building coral Montipora capitata.

Parental effects are ubiquitous in nature and in many organisms play a particularly critical role in the transfer of symbionts across generations; however, their influence and relative importance in the marine environment has rarely been considered. Coral reefs are biologically diverse and productive marine ecosystems, whose success is framed by symbiosis between reef-building corals and unicellular dinoflagellates in the genus Symbiodinium. Many corals produce aposymbiotic larvae that are infected by Symbiodinium from the environment (horizontal transmission), which allows for the acquisition of new endosymbionts (different from their parents) each generation. In the remaining species, Symbiodinium are transmitted directly from parent to offspring via eggs (vertical transmission), a mechanism that perpetuates the relationship between some or all of the Symbiodinium diversity found in the parent through multiple generations. Here we examine vertical transmission in the Hawaiian coral Montipora capitata by comparing the Symbiodinium ITS2 sequence assemblages in parent colonies and the eggs they produce. Parental effects on sequence assemblages in eggs are explored in the context of the coral genotype, colony morphology, and the environment of parent colonies. Our results indicate that ITS2 sequence assemblages in eggs are generally similar to their parents, and patterns in parental assemblages are different, and reflect environmental conditions, but not colony morphology or coral genotype. We conclude that eggs released by parent colonies during mass spawning events are seeded with different ITS2 sequence assemblages, which encompass phylogenetic variability that may have profound implications for the development, settlement and survival of coral offspring.

Variation in Symbiodinium ITS2 sequence assemblages among coral colonies.

Endosymbiotic dinoflagellates in the genus Symbiodinium are fundamentally important to the biology of scleractinian corals, as well as to a variety of other marine organisms. The genus Symbiodinium is genetically and functionally diverse and the taxonomic nature of the union between Symbiodinium and corals is implicated as a key trait determining the environmental tolerance of the symbiosis. Surprisingly, the question of how Symbiodinium diversity partitions within a species across spatial scales of meters to kilometers has received little attention, but is important to understanding the intrinsic biological scope of a given coral population and adaptations to the local environment. Here we address this gap by describing the Symbiodinium ITS2 sequence assemblages recovered from colonies of the reef building coral Montipora capitata sampled across Kane'ohe Bay, Hawai'i. A total of 52 corals were sampled in a nested design of Coral Colony(Site(Region)) reflecting spatial scales of meters to kilometers. A diversity of Symbiodinium ITS2 sequences was recovered with the majority of variance partitioning at the level of the Coral Colony. To confirm this result, the Symbiodinium ITS2 sequence diversity in six M. capitata colonies were analyzed in much greater depth with 35 to 55 clones per colony. The ITS2 sequences and quantitative composition recovered from these colonies varied significantly, indicating that each coral hosted a different assemblage of Symbiodinium. The diversity of Symbiodinium ITS2 sequence assemblages retrieved from individual colonies of M. capitata here highlights the problems inherent in interpreting multi-copy and intra-genomically variable molecular markers, and serves as a context for discussing the utility and biological relevance of assigning species names based on Symbiodinium ITS2 genotyping.

Defining Boundaries for Ecosystem-Based Management: A Multispecies Case Study of Marine Connectivity across the Hawaiian Archipelago.

Determining the geographic scale at which to apply ecosystem-based management (EBM) has proven to be an obstacle for many marine conservation programs. Generalizations based on geographic proximity, taxonomy, or life history characteristics provide little predictive power in determining overall patterns of connectivity, and therefore offer little in terms of delineating boundaries for marine spatial management areas. Here, we provide a case study of 27 taxonomically and ecologically diverse species (including reef fishes, marine mammals, gastropods, echinoderms, cnidarians, crustaceans, and an elasmobranch) that reveal four concordant barriers to dispersal within the Hawaiian Archipelago which are not detected in single-species exemplar studies. We contend that this multispecies approach to determine concordant patterns of connectivity is an objective and logical way in which to define the minimum number of management units and that EBM in the Hawaiian Archipelago requires at least five spatially managed regions.

Ecomorph or endangered coral? DNA and microstructure reveal hawaiian species complexes: Montipora dilatata/flabellata/turgescens & M. patula/verrilli.

M. dilatata, M. flabellata, and M. patula and 80 other scleractinian corals were petitioned to be listed under the US Endangered Species Act (ESA), which would have major conservation implications. One of the difficulties with this evaluation is that reproductive boundaries between morphologically defined coral species are often permeable, and morphology can be wildly variable. We examined genetic and morphological variation in Hawaiian Montipora with a suite of molecular markers (mitochondrial: COI, CR, Cyt-B, 16S, ATP6; nuclear: ATPsß, ITS) and microscopic skeletal measurements. Mitochondrial markers and the ITS region revealed four distinct clades: I) M. patula/M. verrilli, II) M. cf. incrassata, III) M. capitata, IV) M. dilatata/M. flabellata/M. cf. turgescens. These clades are likely to occur outside of Hawai'i according to mitochondrial control region haplotypes from previous studies. The ATPsß intron data showed a pattern often interpreted as resulting from hybridization and introgression; however, incomplete lineage sorting may be more likely since the multicopy nuclear ITS region was consistent with the mitochondrial data. Furthermore, principal components analysis (PCA) of skeletal microstructure was concordant with the mitochondrial clades, while nominal taxa overlapped. The size and shape of verrucae or papillae contributed most to identifying groups, while colony-level morphology was highly variable. It is not yet clear if these species complexes represent population-level variation or incipient speciation (CA<1MYA), two alternatives that have very different conservation implications. This study highlights the difficulty in understanding the scale of genetic and morphological variation that corresponds to species as opposed to population-level variation, information that is essential for conservation and for understanding coral biodiversity.