Lindsay Sarah J, Bonfield James K, Hurles Matthew E
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
Nucleic Acids Res. 2005 Oct 12;33(18):e152. doi: 10.1093/nar/gni152.
Haplotypic sequences contain significantly more information than genotypes of genetic markers and are critical for studying disease association and genome evolution. Current methods for obtaining haplotypic sequences require the physical separation of alleles before sequencing, are time consuming and are not scaleable for large surveys of genetic variation. We have developed a novel method for acquiring haplotypic sequences from long PCR products using simple, high-throughput techniques. This method applies modified shotgun sequencing protocols to sequence both alleles concurrently, with read-pair information allowing the two alleles to be separated during sequence assembly. Although the haplotypic sequences can be assembled manually from the resultant data using pre-existing sequence assembly software, we have devised a novel heuristic algorithm to automate assembly and remove human error. We validated the approach on two long PCR products amplified from the human genome and confirmed the accuracy of our sequences against full-length clones of the same alleles. This method presents a simple high-throughput means to obtain full haplotypic sequences potentially up to 20 kb in length and is suitable for surveying genetic variation even in poorly-characterized genomes as it requires no prior information on sequence variation.
单倍型序列包含的信息比遗传标记的基因型多得多，对于研究疾病关联和基因组进化至关重要。目前获取单倍型序列的方法需要在测序前对等位基因进行物理分离，耗时且无法扩展用于大规模遗传变异调查。我们开发了一种使用简单的高通量技术从长PCR产物中获取单倍型序列的新方法。该方法应用改良的鸟枪法测序方案同时对两个等位基因进行测序，读对信息允许在序列组装过程中分离两个等位基因。虽然可以使用现有的序列组装软件从所得数据中手动组装单倍型序列，但我们设计了一种新颖的启发式算法来实现自动组装并消除人为误差。我们对从人类基因组扩增的两个长PCR产物验证了该方法，并对照相同等位基因的全长克隆确认了我们序列的准确性。这种方法提供了一种简单的高通量手段来获得长度可能高达20 kb的完整单倍型序列，并且适用于即使在特征不明确的基因组中进行遗传变异调查，因为它不需要关于序列变异的先验信息。
