Single haplotype assembly of the human genome from a hydatidiform mole

Abstract

A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can improve assembly, even the longest available reads do not resolve all regions. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly.

Figure 1.

Comparison of contig count and contig N50 between CHM1_1.1 (GenBank GCA_000306695.2) and HuRef (J. Craig Venter assembly; GenBank GCA_000002125.2), ALLPATHS (GenBank AEKP00000000.1) and YH_2.0 (GenBank GCA_000004845.2) WGS assemblies. CHM1_1.1 has only 10%–20% of the number of total contigs as the other assemblies and has a contig N50 1.5 to six times larger.

Figure 2.

(A) WGS assembly from the first pass (CHM1_1.0; GCF_000306695.1, bronze line) on Chromosome 1p12 (NC_018912.1: 121,050,000-121,400,000) demonstrated a gap (gray box) in the assembly (assembly name: AMYH010000980.1, green lines). Using MEGABLAST, two CH17 clones (AC247039.2 and AC253572.3, red lines) aligned to the region and appeared to span the gap. (B) By incorporating these BAC sequences into the assembly, the gap was subsequently resolved in CHM1_1.1 (NC_018912.2: 121,050,000–121,650,000). The tiling path components, FP325311.11, AC241952.2, AC247039.2, AC253572.3, and AC241377.3 indicate the clone names used to resolve the gap. The clones from A are indicated in red while the other clones are in purple. The final assembly–assembly alignment is indicated in purple, showing the gap resolution.

Figure 3.

(A) Comparison of segmental duplications (SDs) in GRCh37 and CHM1.1 assemblies predicted by WGAC analysis by chromosome. The duplication content is comparable between GRCh37 and CHM1_1.1 assemblies indicating good assembly quality. (B) Venn diagram of SDs in GRCh37 and CHM1_1.1 assemblies shows that most duplications are shared between the assemblies.

Figure 4.

Functional consequences of CHM1 heterozygous variants not in repetitive sequence (HNR variants). Approximately 97% of HNR variants are intergenic or intronic. Of the remaining 3% of other variants, ∼48% are in the 3′ or 5′ UTR, 17% are silent, and 35% are coding (missense, nonsense, essential splice site).

Figure 5.

Overview of the Chr 11 (NC_018922.2) 1.9-Mb region, exhibiting three alignment bins with a large number of PacBio “cliff” reads where the alignment coverage dropped off sharply. WGS component (light green lines) boundaries flanked by such reads are marked with red dashed lines. The ends of each component at the boundary are labeled with letters to show orientation. Pairs of alignments corresponding to three different PacBio reads are marked in yellow, green, and dark blue. These alignments overlap by < 10% on each of the reads. The split alignments for these three reads suggest that the two WGS components marked in purple should be inverted and translocated as indicated by the arrow at the top of the image. The other PacBio reads in these bins exhibit the same pattern of split alignments, which supports the proposed reordering and orientation of the WGS components. The bottom light green lines show a proposed tiling path with the orientation corrected; the letters indicate where each end of the initial tiling path components should be placed.