The human reference sequence has provided a foundation for studies of genome structure, human variation, evolutionary biology, and disease. At the time the reference was originally completed there were some loci recalcitrant to closure; however, the degree to which structural variation and diversity affected our ability to produce a representative genome sequence at these loci was still unknown. Many of these regions in the genome are associated with large, repetitive sequences and exhibit complex allelic diversity such producing a single, haploid representation is not possible. To overcome this challenge, we have sequenced DNA from two hydatidiform moles (CHM1 and CHM13), which are essentially haploid. CHM13 was sequenced with the latest PacBio technology (P6-C5) to 52X genome coverage and assembled using Daligner and Falcon v0.2 (GCA_000983455.1, CHM13_1.1). Compared to the first mole (CHM1) PacBio assembly (GCA_001007805.1, 54X) contig N50 of 4.5Mb, the contig N50 of CHM13_1.1 is almost 13Mb, and there is a 13-fold reduction in the number of contigs. This demonstrates the improved contiguity of sequence generated with the new chemistry. We annotated 50,188 RefSeq transcripts of which only 0.63% were split transcripts, and the repetitive and segmental duplication content was within the expected range. These data all indicate an extremely high quality assembly. Additionally, we sequenced CHM13 DNA using Illumina SBS technology to 60X coverage, aligned these reads to the GRCh37, GRCh38, and CHM13_1.1 assemblies and performed variant calling using the SpeedSeq pipeline. The number of single nucleotide variants (SNV) and indels was comparable between GRCh37 and GRCh38. Regions that showed increased SNV density in GRCh38 compared to GRCh37 could be attributed to the addition of centromeric alpha satellite sequence to the reference assembly. Alternatively, regions of decreased SNV density in GRCh38 were concentrated in regions that were improved from BAC based sequencing of CHM1 such as 1p12 and 1q21 containing the SRGAP2 gene family. The alignment of PacBio reads to GRCh37 and GRCh38 assemblies allowed us to resolve complex loci such as the MHC region where the best alignment was to the DBB (A2-B57-DR7) haplotype. Finally, we will discuss how combining the two high quality mole assemblies can be used for benchmarking and novel bioinformatics tool development.
Organization: McDonnell Genome Institute at Washington University