This guest post is by Karyn Meltz Steinberg on her preprint (with coauthors) Single haplotype assembly of the human genome from a hydatidiform mole, bioRxived here.

The human reference sequence is a mosaic of many DNA sources patched together to create the (mostly) contiguous chromosomes we all use every day in genomics labs. This mélange of haplotypes can result in reference representations that do not exist. For example, in GRCh37 at the MRC1 locus mixed haplotypes led to the presence of two gene models that represent false duplications and a gap that affected alignments of short reads. The problem of diploid source DNA was even worse in regions of structural variation where it was difficult to distinguish allelic variation from paralogous variation. The assembly structure in these regions was often wrong with either a collapsed assembly leading to missing sequence or with haplotype expanse, meaning 2, 3 or more haplotypes were represented on the chromosome sequence. The genomic resources associated with the essentially haploid complete hydatidiform mole, CHM1, have opened the door to allow us to address these issues.

What is essentially haploid? Well, what usually happens is that a sperm (usually bearing an X chromosome) fertilizes an egg that doesn’t have a nucleus (thus no DNA), the sperm DNA doubles, giving the correct chromosome complement, but with both pairs of chromosomes being identical. This cell divides and grows but does not form a normal embryo. In the early 1990s, Dr. Urvashi Surti was able to make an immortalized cell line from CHM1 tissue using hTERT. She karyotyped each passage to check that it maintained ploidy and that there were no gross somatic rearrangements.

Dr. Pieter de Jong then created an indexed BAC library from this high fidelity material. These BACs could then be used to resolve structurally complex regions such as 17q21.311. We continued working on sequencing more tiling paths across structurally complex regions; however, it was not practical or cost-efficient to Sanger sequence every single clone in the BAC library. As work continued, it became clear that developing the Primary Assembly using a single haplotype resource could be very powerful. This was possible due to the efforts of the Genome Reference Consortium (GRC) to extend the assembly model to include more than one sequence representation for a give region. We used Illumina sequencing technology and a reference based assembly algorithm developed at NCBI to produce an initial assembly. We then integrated the BAC sequences into the assembly to improve regions that are nearly impossible to assembly using whole genome strategies. The result is the highest quality whole genome sequence human genome assembly that is publicly available to date as assessed by metrics including contig and scaffold N50, repetitive element content and gene annotation.

So what–how can this help us, you ask? For the first time, one single haplotype of the human genome is represented. The fact that CHM1 is haploid means that we are able to finally go into the messy regions of the genome and resolve the genomic architecture as well as put any structural variation in the context of surrounding linked allelic variation. These are often biologically interesting regions; for example genes related to immune response and metabolism that are probably associated with complex traits are usually members of large gene families in segmentally duplicated sequence.

A fine example of the power of this haploid resource is also on BioRxiv, “Sequencing of the human IG light chain loci from a hydatidiform mole BAC library reveals locus-specific signatures of genetic diversity” (disclosure: this is also co-authored by me). The IG light chain genes encode for one part of immunoglobulin molecules that are expressed by B cells in response to antigenic stimulation (the heavy chain, IGH, was also resolved using CHM1 resources last year2). They are part of large gene families formed by duplication at three loci in the human genome. In previous versions of the reference assembly, these loci were comprised of sequence from multiple DNA sources that may have undergone somatic rearrangement. By sequencing BAC clones in a tiling path across these loci we now have a single haplotype representation of germline DNA sequence that allows us to perform accurate analyses of variation.

REFERENCES

1 Itsara, A. et al. Resolving the breakpoints of the 17q21.31 microdeletion syndrome with next-generation sequencing. American journal of human genetics 90, 599-613, doi:10.1016/j.ajhg.2012.02.013 (2012).
2 Watson, C. T. et al. Complete haplotype sequence of the human immunoglobulin heavy-chain variable, diversity, and joining genes and characterization of allelic and copy-number variation. American journal of human genetics 92, 530-546 (2013).