De novo assembly of a haplotype-resolved human genome

Abstract

The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.

De novo assembly and phasing of a Korean human genome

“…Phasing was performed with PacBio long reads, Illumina short reads, 10X Genomics linked reads 4 (30× ), and reads from BACs representing a single haplotype (47× ). Heterozygous SNVs called from these methods are unambiguously assigned to two alternative phases, producing phased blocks with an N50 length of 11.6 Mb, considerably longer than previously reported 4,6,8,15,16 (Table 1). We assessed the accuracy of the phased blocks against the end sequences of BACs, and found a long-range switch error rate to be under 0.3%.…”

“…We were able to validate 271 out of 276 SVs with BAC contigs generated by SMRT sequencing (Supplementary Table 12). Compared to previous studies 6,[8][9][10][11] , a total of 11,927 variants were previously unreported, which account for approximately 47% (3,465) and 76% (7,710) of all deletions and insertions, respectively ( Fig. 2a and Extended Data Fig.…”

“…The final assembly after polishing with Illumina reads (Extended Data Fig. 4a) is characterized by marked contiguity that has not been achieved by non-reference assemblies of the human diploid genome [6][7][8] so far, and improves on the previous best 6 N50 length by 18 Mb ( Table 1). The largest 91 scaffolds, for example, cover 90% of the genome and 8 chromosomal arms are spanned by single scaffolds (Fig.…”

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly

“…The reference assembly is not just a substrate for alignment, but is also the coordinate system on which we annotate our biological knowledge. Several recently published individual human de novo assemblies have been favorably compared to GRCh38 with respect to continuity metrics, and although they each contain sequence not present in the reference assembly, none yet surpass the global quality of GRCh38 Steinberg et al 2014;Berlin et al 2015;Cao et al 2015;Pendleton et al 2015;Seo et al 2016;Shi et al 2016). Such assemblies are often suggested as sequence sources for use in closure of reference assembly gaps, whereas other studies have called for one or more individual genomes to replace the reference .…”

Direct determination of diploid genome sequences

“…In a few cases, these diploid de novo assemblies have been demonstrated for small and midsized genomes (Jones et al 2004;Chin et al 2016). There are two extant instances of diploid de novo assemblies of human genomes-one obtained by Sanger sequencing of multiple libraries (Levy et al 2007), and one from thousands of separate clone pools, each representing a small, low-throughput partition of the genome (Cao et al 2015).…”