Toehold probe-based interrogation for haplotype phasing of long nucleic acid strands

The arrangement of multiple single nucleotide polymorphisms (SNPs) in a gene, called a haplotype phase, is increasingly recognized as critical for accurate determination of disease risk and severity.

Determination of complete chromosomal haplotypes by bulk DNA sequencing

Haplotype phase represents the collective genetic variation between homologous chromosomes and is an essential feature of non-haploid genomes. Determining the haplotype phase requires knowledge of both the genotypes at variant sites and their linkage across each chromosome. Haplotype linkage can be either inferred statistically from a genotyped population, or determined by long-range sequencing of an individual genome. However, extending haplotype inference to the whole-chromosome scale remains challenging and usually requires special experimental techniques. Here we describe a general computational strategy to determine complete chromosomal haplotypes using a combination of bulk long-range sequencing and Hi-C sequencing. We demonstrate that this strategy can resolve the haplotypes of parental chromosomes in diploid human genomes at high precision (99%) and completeness (98%), and is further able to assemble the syntenic organization of aneuploid genomes (“digital karyotype”).

trio-sga: facilitating de novo assembly of highly heterozygous genomes with parent-child trios

MotivationMost DNA sequence in diploid organisms is found in two copies, one contributed by the mother and the other by the father. The high density of differences between the maternally and paternally contributed sequences (heterozygous sites) in some organisms makes de novo genome assembly very challenging, even for algorithms specifically designed to deal with these cases. Therefore, various approaches, most commonly inbreeding in the laboratory, are used to reduce heterozygosity in genomic data prior to assembly. However, many species are not amenable to these techniques.ResultsWe introduce trio-sga, a set of three algorithms designed to take advantage of mother-father-offspring trio sequencing to facilitate better quality genome assembly in organisms with moderate to high levels of heterozygosity. Two of the algorithms use haplotype phase information present in the trio data to eliminate the majority of heterozygous sites before the assembly commences. The third algorithm is designed to reduce sequencing costs by enabling the use of parents’ reads in the assembly of the genome of the offspring. We test these algorithms on a ‘simulated trio’ from four hap-loid datasets, and further demonstrate their performance by assembling three highly heterozygous Heliconius butterfly genomes. While the implementation of trio-sga is tuned towards Illumina-generated data, we note that the trio approach to reducing heterozygosity is likely to have cross-platform utility for de novo assembly.