Genomic Characterization of Large Heterochromatic Gaps in the Human Genome Assembly

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Abstract
The largest gaps in the human genome assembly correspond to multi-megabase heterochromatic regions composed primarily of two related families of tandem repeats, Human Satellites 2 and 3 (HSat2,3). The abundance of repetitive DNA in these regions challenges standard mapping and assembly algorithms, and as a result, the sequence composition and potential biological functions of these regions remain largely unexplored. Furthermore, existing genomic tools designed to predict consensus-based descriptions of repeat families cannot be readily applied to complex satellite repeats such as HSat2,3, which lack a consistent repeat unit reference sequence. Here we present an alignment-free method to characterize complex satellites using whole-genome shotgun read datasets. Utilizing this approach, we classify HSat2,3 sequences into fourteen subfamilies and predict their chromosomal distributions, resulting in a comprehensive satellite reference database to further enable genomic studies of heterochromatic regions. We also identify 1.3 Mb of non-repetitive sequence interspersed with HSat2,3 across 17 unmapped assembly scaffolds, including eight annotated gene predictions. Finally, we apply our satellite reference database to high-throughput sequence data from 396 males to estimate array size variation of the predominant HSat3 array on the Y chromosome, confirming that satellite array sizes can vary between individuals over an order of magnitude (7 to 98 Mb) and further demonstrating that array sizes are distributed differently within distinct Y haplogroups. In summary, we present a novel framework for generating initial reference databases for unassembled genomic regions enriched with complex satellite DNA, and we further demonstrate the utility of these reference databases for studying patterns of sequence variation within human populations. At least 5–10% of the human genome remains unassembled, unmapped, and poorly characterized. The reference assembly annotates these missing regions as multi-megabase heterochromatic gaps, found primarily near centromeres and on the short arms of the acrocentric chromosomes. This missing fraction of the genome consists predominantly of long arrays of near-identical tandem repeats called satellite DNA. Due to the repetitive nature of satellite DNA, sequence assembly algorithms cannot uniquely align overlapping sequence reads, and thus satellite-rich domains have been omitted from the reference assembly and from most genome-wide studies of variation and function. Existing methods for analyzing some satellite DNAs cannot be easily extended to a large portion of satellites whose repeat structures are complex and largely uncharacterized, such as Human Satellites 2 and 3 (HSat2,3). Here we characterize HSat2,3 using a novel approach that does not depend on having a well-defined repeat structure. By classifying genome-wide HSat2,3 sequences into subfamilies and localizing them to chromosomes, we have generated an initial HSat2,3 genomic reference, which serves as a critical foundation for future studies of variation and function in these regions. This approach should be generally applicable to other classes of satellite DNA, in both the human genome and other complex genomes.

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