Towards complete and error-free genome assemblies of all vertebrate species

Abstract

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 species1–4. To address this issue, the international Genome 10K (G10K) consortium5,6 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 Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries.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.

Complete vertebrate mitogenomes reveal widespread repeats and gene duplications

“…The VGP version 1 assembly pipeline developed for the nuclear genome uses Continuous Long Reads (CLR) and the Pacific Biosciences (PacBio) assembler FALCON to generate contigs [35,36]. When initially inspecting the contigs, we noted the absence of contigs representing the mitogenome in 75% of species.…”

“…The high level of similarity is supportive of the overall quality of the mitoVGP assembly, which is also confirmed by its Q44.30 base call accuracy and 100% identity to the NOVOPlasty assembly in non-repetitive regions. In the kakapo (Strigops habroptilus), an entire 2.3-kbp CR region, including a~925-bp-long repeat (repeat unit = 84 bp), was also missing from the RefSeq sequence (long-range PCR and direct Sanger sequencing [35,43], Fig. 2c).…”

“…Among these assemblies, 8 showed duplications and/or large repetitive elements. These include 5 birds belonging to 5 separate orders that have highly similar, but not identical patterns of duplicated genes in the CR (MT-CYB, MT-TT, MT-TP, MT-ND6 and MT-TE): the Anna's hummingbird (Calypte anna) [35], red-legged seriema (Cariama cristata), Swainson's thrush (Catharus ustulatus), maguari stork (Ciconia maguari), whiskered treeswift (Hemiprocne comata), and European golden plover (Pluvialis apricaria). For example, in the maguari stork (Ciconia maguari, Fig.…”

“…Here, we describe a new mitochondrial genome assembly pipeline (mitoVGP) developed in the framework of the Vertebrate Genomes Project (VGP) (www. vertebrategenomesproject.org) [34,35]. The VGP aims to generate near complete and error-free reference genome assemblies representing all vertebrate species, and in its Phase 1, it is currently targeting one species for each vertebrate order.…”

“…The VGP aims to generate near complete and error-free reference genome assemblies representing all vertebrate species, and in its Phase 1, it is currently targeting one species for each vertebrate order. Our mitoVGP pipeline complements the nuclear assembly VGP pipeline [35], which has become a standard approach for many species, by combining long reads for structural accuracy and short reads for base calling accuracy. We applied mitoVGP to determine the full mitogenome sequence of 100 vertebrate species, including 33 species for which a reference mtDNA sequence was not previously available.…”