Large scale molecular haplotyping using next generation sequencing

测序整个基因组的成本正在迅速下降，1000只动物和人类S的基因组正在进行测序。大多数哺乳动物的基因组中每条染色体都有两个拷贝。每条染色体的两个副本非常相似，但仍然包含数万个微小的差异。这些差异大多是构成染色体的化学碱基序列的单一变化。目前的测序方法不能区分两条染色体的序列，而公布的“完整”序列实际上是每个染色体对成员的单一组合。这掩盖了这样一个事实，即每个基因都有两种略有不同的形式，每条染色体上有一种，这两种不同的形式被称为等位基因。了解单个染色体的序列可以使许多其他研究变得容易得多。每条染色体包含一组作为一个块遗传的多个基因，知道一个基因的等位基因就可以预测与之遗传的其他基因的等位基因，从而可以更轻松地预测基因组中所有基因的等位基因。此外，基因主要由同一染色体上的相邻区域控制，导致不同区域之间的相互作用，知道哪些等位基因彼此相邻，可以更容易地识别这些相互作用。我们将开发两种方法来识别每个染色体的大区域序列。首先，我们将提取只包含每个染色体的单一拷贝的单个精子或卵母细胞，其次，我们将稀释从多个细胞或单个细胞制备的DNA，直到出现来自每个染色体相同区域的DNA片段的可能性很小。然后，我们将使用为大量DNA开发的标准方法来扩增DNA，并使用利物浦基因组研究中心的下一代基因组测序仪获得DNA序列。我们将通过对一头精英公牛进行测序来初步测试这两种方法，这头公牛的后代已经由曾傑瑞·泰勒教授进行了大量的基因分型，泰勒教授是该项目的指定合作者。这将使我们能够根据基于已知亲缘关系和大量基因数据的成熟方法确定的单倍型来验证我们的单倍型。东非奶牛是澳大利亚新英格兰大学的约翰·吉布森进行的一项重大遗传学研究的对象，他是该项目的指定合作者。这项研究正在开发测试，以确定该地区最好的奶牛，以便选择它们进行进一步饲养。这包括用超过50万个标记对2000只这些动物进行基因分型，以确定每种动物在每个位置携带的等位基因。然而，如上所述，这种方法不能识别染色体上的单倍型。我们将使用我们在项目第一部分开发的最合适的单分子测序方法，通过对这个群体中100只动物基因组的五分之一进行测序，来发现这个群体中常见的单倍型。然后，我们将结合另一项研究中获得的2000只动物的基因数据和本研究中100只动物获得的常见单倍型来重建种群中所有2000只动物的单倍型。这些数据将使未来进行全基因组协会的研究成为可能，以确定与该人群中的特定特征相关的基因，如产奶量或抗病能力。最后，我们将开发软件来自动化我们的大部分分析，以便其他工作人员可以轻松地使用我们开发的方法。

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.