An efficient method for multi-locus molecular haplotyping

Abstract

Many methods exist for genotyping--revealing which alleles an individual carries at different genetic loci. A harder problem is haplotyping--determining which alleles lie on each of the two homologous chromosomes in a diploid individual. Conventional approaches to haplotyping require the use of several generations to reconstruct haplotypes within a pedigree, or use statistical methods to estimate the prevalence of different haplotypes in a population. Several molecular haplotyping methods have been proposed, but have been limited to small numbers of loci, usually over short distances. Here we demonstrate a method which allows rapid molecular haplotyping of many loci over long distances. The method requires no more genotypings than pedigree methods, but requires no family material. It relies on a procedure to identify and genotype single DNA molecules, and reconstruction of long haplotypes by a 'tiling' approach. We demonstrate this by resolving haplotypes in two regions of the human genome, harbouring 20 and 105 single-nucleotide polymorphisms, respectively. The method can be extended to reconstruct haplotypes of arbitrary complexity and length, and can make use of a variety of genotyping platforms. We also argue that this method is applicable in situations which are intractable to conventional approaches.

Figure 1

Principle of the method. Diploid cells (a) are shown containing two haplotypes (upper-case, blue; and lower-case, yellow). DNA is prepared (b) with inevitable breakage through shearing, and dispensed at extreme dilution into aliquots (c), each containing much less than a complete genome. Initially, the samples are pre-screened by PCR to find out which loci they each contain, but without genotyping (d) red circles indicate a positive PCR result; red lines show the inferred extent of the fragments in each aliquot. Only a handful of aliquots then need to be genotyped for each locus (e). From these results, the complete haplotypes (f) can be reconstructed. Note that a few aliquots may contain mixed haplotypes. In this case, two of the aliquots give the partial haplotypes rs and RS, whilst the rightmost one gives the mixed haplotype rS—the correct linkage phase is inferred from the majority (in this case, rs/RS rather than rS/Rs).

Figure 2

Pre-screening, genotyping and haplotype reconstruction for X-chromosome markers. (a) Pre-screening results. The 94 sub-genomic aliquots are arranged from left to right, and the 20 X-chromosome loci are represented from top to bottom (spacing reflects genomic locations). Red dots indicate which loci are present in each aliquot (green dots = uncertain), and red/green lines represent the DNA fragments inferred to lie in each aliquot. Grey shading indicates the aliquots and loci which were selected for genotyping. (b) Genotyping results for the selected aliquots and loci. Letters show the allele found; M = mixed genotyping result; ? = no genotype (failed reaction). Two distinct haplotypes (blue and yellow) can be reconstructed. Each genotyping result is coloured to indicate which haplotype it is inferred to originate. Green indicates that the haplotype origin for that data point cannot be inferred, either because the locus is not heterozygous or because it gives a mixed (‘M’) genotype result. The two reconstructed haplotypes (denoted 1 and 2) are indicated; heterozygous loci in bold. On the right are shown the calculated log odds supporting the haplotypes for each step between consecutive heterozygous loci.

Figure 3

Genotyping and haplotype reconstruction for chromosome 21 markers. Initial PCR pre-screening results for the 96 aliquots are not shown: only the genotyping results for the selected aliquots and loci are shown (format and notation as for Figure 2b).