High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species^1–4. To address this issue, the international Genome 10K (G10K) consortium^5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

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).

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.

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.

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.