Chromosome-Range Whole-Genome High-Throughput Experimental Haplotyping by Single-Chromosome Microdissection

Abstract

Haplotype is fundamental genetic information; it provides essential information for deciphering the functional and etiological roles of genetic variants. As haplotype information is closely related to the functional and etiological impact of genetic variants, it is widely anticipated that haplotype information will be extremely valuable in a wide spectra of applications, including academic research, clinical diagnosis of genetic disease and in the pharmaceutical industry. Haplotyping is essential for LD (linkage disequilibrium) mapping, functional studies on cis-interactions, big data imputation, association studies, population studies, and evolutionary studies. Unfortunately, current sequencing technologies and genotyping arrays do not routinely deliver this information for each individual, but yield only unphased genotypes. Here, we describe a high-throughput and cost-effective experimental protocol to obtain high-resolution chromosomal haplotypes of each individual diploid (including human) genome by the single-chromosome microdissection and sequencing approach.

Keywords: Chromosome-length; Experimental; Haplotype; High-throughput; Whole-genome.

Fig. 1

Difference between genotypes and haplotypes

Fig. 2

An illustration that haplotypes rather than genotypes are functionally relevant. In this special case, two different individuals have the same genotypes (G/T and C/A) but different haplotypes at two SNP loci. SNP-1 (G/T) occurred in an enhancer (G/T) that is essential for the expression of this gene, in which Allele-T is a null allele. SNP-2 (C/A) resides in an exon of this gene, in which Allele-A will disrupt the translation with an early stop codon. The production of this protein requires a cis-relatlonship between the enhancer and exon. Thus, Person-1 can produce this protein because one of his gene copies contains both functional alleles on the same chromosome (cis); Person-2 cannot produce this protein because neither of his gene copies contains two functional alleles. Please note that the enhancers may be close to a gene, or as far as 1 million base away from its regulatory target

Fig. 3

The pipeline of haplotype determination described in this chapter. In this pipeline, we will first culture the cells to metaphase and then isolate single-chromosomes; the isolated single-chromosomes will be then subjected to whole-genome amplification (WGA) followed by high-throughput sequencing; the sequencing data will be computationally analyzed (removing the noises, increasing the resolution and output the haplotypes)

Fig. 4

A chromosome is being microdissected by a laser beam directed by a computer