Large scale molecular haplotyping using next generation sequencing

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The cost of sequencing whole genomes is falling rapidly and the genomes of 1000's of animals and humans are being sequenced. Most mammalian genomes contain two copies of each chromosome. The two copies of each chromosome are very similar but nevertheless contain tens of thousands of small differences. These differences are mostly single changes in the sequence of chemical bases that compose the chromosome. Present sequencing methods cannot discriminate between the sequences of the two chromosomes and the 'complete' sequences that are published are in fact a single composite of the members of each chromosome pair. This disguises the reality that each gene has two slightly different forms, one on each chromosome, the two different forms are known as alleles. Knowing the sequences of the individual chromosomes could make many other studies much easier. Each chromosome contains groups of multiple genes which are inherited as a block, knowing the allele of one gene makes it possible to predict the alleles of the others that are inherited with it, making it possible to predict the alleles of all genes in the genome with much less effort. Also genes are mainly controlled by adjacent regions on the same chromosome leading to interactions between different regions, knowing which alleles are adjacent to each other makes it much easier to identify these interactions. We will develop two methods for identifying the sequences of large regions of each chromosome. Firstly we will take single sperm or oocytes which only contain a single copy of each chromosome and secondly we will dilute DNA prepared from many cells or a single cell until there is only a small chance that fragments of DNA from the same region of each chromosome are present. We will then amplify the DNA using standard methods developed for larger quantities of DNA and obtain the sequence of the DNA using the next generation genome sequencers at the Liverpool Center for Genomic Research. We will initially test the two methods by sequencing an elite bull that has large numbers of offspring that have already been genotyped by Prof Jerry Taylor who is a named collaborator on this project. This will enable us to validate our haplotypes against those identified by well established methods based on known parentage and large amounts of genotype data. East African dairy cattle are the subject of a major genetic study by John Gibson of the University of New England in Australia who is a named collaborator on this project. That study is developing tests to identify the best dairy cattle in the region so that they can be selected for further breeding. This involves genotyping 2000 of these animals with over 500,000 markers to identify the alleles carried by each animal at each position. However as noted above this method cannot identify the haplotypes along a chromosome. We will use the most appropriate single molecule sequencing methods that we have developed in the first part of the project to discover the common haplotypes in this population by sequencing a fifth of the genome of 100 animals from this population. We will then combined the genotype data from the 2000 animals obtained in the other study with the common haplotypes obtained from the 100 animals in this study to reconstruct the haplotypes of all 2000 animals in the population. This data will make it possible to undertake a future Genome Wide Association Study to identify genes that are associated with specific traits such as milk production or disease resistance in this population. Finally we will develop software to automate most of our analysis so that other workers can easily use the methods that we have developed.