Semi-automated assembly of high-quality diploid human reference genomes

Abstract

The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has benefitted society1,2. However, it still has many gaps and errors, and does not represent a biological genome as it is a blend of multiple individuals3,4. Recently, a high-quality telomere-to-telomere reference, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a nearly homozygous genome5. To address these limitations, the Human Pangenome Reference Consortium formed with the goal of creating high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and assembly approaches yield the most complete and accurate diploid genome assembly with minimal manual curation. Approaches that used highly accurate long reads and parent–child data with graph-based haplotype phasing during assembly outperformed those that did not. Developing a combination of the top-performing methods, we generated our first high-quality diploid reference assembly, containing only approximately four gaps per chromosome on average, with most chromosomes within ±1% of the length of CHM13. Nearly 48% of protein-coding genes have non-synonymous amino acid changes between haplotypes, and centromeric regions showed the highest diversity. Our findings serve as a foundation for assembling near-complete diploid human genomes at scale for a pangenome reference to capture global genetic variation from single nucleotides to structural rearrangements.

Pushing the limits of HiFi assemblies reveals centromere diversity between two Arabidopsis thaliana genomes

“…On the other hand, HiFi-FALCON, HiFi-HiCanu and HiFi-Hifiasm nuclear scaffolds contained very similar amounts of 5S rDNA arrays, 1.64–1.68 Mb, and of centromeres, 13.63–13.69 Mb. To investigate the reliability of our assemblies in these repetitive regions, we analyzed potentially collapsed and expandable sequences in the scaffolded assemblies ( 67 , 73 ). According to annotations of repeat features in the assemblies, 5S rDNAs and centromeres did not appear to contribute substantially to the collapsed sequences in the HiFi-FALCON, HiFi-HiCanu and HiFi-Hifiasm nuclear scaffolds ( Supplementary Figure S6 ).…”

A refined characterization of large-scale genomic differences in the first complete human genome

“…It is anticipated that the T2T Consortium will generate more complete genome assemblies from a diversity of human samples and non-human primates. These will help us to fully understand the extent of complex/discrepant regions in humans [4][5][6][7][8][9][10][11]25,26 and their biological impact using reference-free approaches.…”

“…2c). We examined the length of CR1 gene in the 94 long-read human genome assembly from the Human Pangenome Reference Consortium (HPRC) [24][25][26] and the length of CR1 in 79 assemblies coincides with that of T2T-CHM13. This suggests that T2T-CHM13 carries the major allele of CR1 (allele frequency: 0.84) (Fig.…”

“…S6). To better characterize the diversity of the KLRC region, we systematically investigated the discrepant region with the 94 long-read genome assemblies from the HPRC dataset [24][25][26] . We found 1 duplication and 11 deletions of KLRC2 in the 94 long-read genome assemblies (Table S7).…”

Long-read genomes reveal pangenomic variation underlying yeast phenotypic diversity

“…In addition to biparental crosses, this strategy can be utilized for linkage mapping studies on other groups of related individuals, such as family studies in humans. As the cost and analytical constraints of generating long-read genome assemblies continues to decline (Jarvis et al 2022), it will become possible to generate high-confidence de novo whole genome sequences of every individual in a mapping study, including genome-wide association studies of unrelated humans. We expect that assembly of highly complete genomes using long-read sequencing will be vital for us to understand the genetic sources of trait variation.…”