Integrated Assembly Software for Sanger and Next Generation Sequence Technologies

描述（由申请人提供）：下一代（Next-gen）测序技术的出现已经开始了全基因组测序和重测序的激增，仅在2008年就有四篇论文描述了五个完整的人类基因组。Knome公司现在甚至使用下一代测序技术为客户提供整个基因组序列。这些发展，再加上基因组的定向重测序，预示着1000美元人类基因组的一天。大规模全人类基因组重测序（WHGR）将对个性化医疗、人类进化和人类多样性等领域产生巨大影响。然而，为了充分实现这一潜力，必须大幅提高软件能力，以应对这些项目产生的大量数据、技术特定数据特征的多样性以及简单分析60亿碱基对二倍体人类基因组所带来的重大挑战。此外，我们看到有一天技术的改进和成本的降低使WHGR像今天的细菌基因组测序一样普遍。要实现这一点，组装和分析软件必须能够被更广泛、更不精通计算机的研究人员所使用，而不是现在解码信息的高度专业化的生物信息学团队。此外，即使对于资金充足的研究实验室来说，计算机资源也比大型测序中心有限得多。因此，该提案的总体目标是开发下一代序列组装和分析管道DESKAPP，其将在可负担得起的（5000美元）高端台式计算机上运行，并在合理的时间范围内（几天，而不是几周）产生人类基因组序列。DESKAPP的WHGR将涉及参考引导的主组装以及从头组装分支，以表征新基因组相对于参考的独特区域。组装体的合并产生了完整的序列，其可以通过基于网络的外部数据库搜索来评估基因内容、单核苷酸多态性（SNP）和结构变异（SV;插入缺失、倒位、易位），以鉴定已知的等位基因变异，并通过直接检查序列来鉴定新的多态性。一个磁盘排序对齐算法允许的数据集，这是太大的内存处理进行评估和集群组装SeqMan N-Gen（SM N-Gen），我们的桌面组装引擎。使用原型DSA-SM N-Gen流水线，我们已经使用DSA在31小时内将来自James沃森基因组的整个7.4x 454数据集处理成布局文件，并且已经使用SM N-Gen组装了三个染色体：8; 21;和X1。组装时间从染色体21的1小时变化到平均大小的染色体（例如染色体8）的10.6小时。总之，这些结果证明了为WHGR构建DESKAPP管道的可行性。第二阶段的目标是在此基础上建立一个无缝的管道，在几天内完成人类基因组的桌面组装和分析。 公共卫生相关性：下一代测序技术通过以不断降低的成本提供前所未有的数量的DNA序列数据，已经开始了整个生物学的新革命。这些数据在个性化医疗的新兴时代和探索生命的巨大多样性方面将是无价的。该项目的目标是开发桌面计算机软件，使任何规模的研究实验室和诊所都能实现这些新技术的承诺。

DESCRIPTION (provided by applicant): The advent of next-generation (Next-gen) sequencing technologies has begun a surge in whole genome sequencing and resequencing, exemplified spectacularly by four papers describing five complete human genomes in 2008 alone. One company, Knome, now even offers customers their entire genome sequence using Next-gen sequencing technology. These developments, together with targeted resequencing of genome, presage the day of the $1000 human genome. Broad-scale whole human genome resequencing (WHGR) will have enormous impact on the areas of personalized medicine, human evolution and human diversity. To fully realize that potential, however, software capabilities must be dramatically enhanced to meet the significant challenges posed by the sheer volume of data generated in these projects, the diversity of technology-specific data characteristics and simply analyzing the 6 billion base pair diploid human genome. Moreover, we see the day when technology improvements and cost reductions make WHGR as commonplace as bacterial genome sequencing has become today. For that to occur, assembly and analysis software must be accessible to a far broader and less computer savvy range of researchers than the highly specialized bioinformatics teams that decode the information now. Also, computer resources are far more limited even for a well funded research laboratory than available to a large sequencing center. Therefore, the overall goal of this proposal is to develop a Next-gen sequence assembly and analysis pipeline, DESKAPP, that will run on an affordable ($5000) high- end desktop computer and produce a human genome sequence in a reasonable timeframe (days, not weeks). WHGR by DESKAPP will involve a reference-guided main assembly as well as a de novo assembly branch to characterize unique regions of the new genome relative to the reference. Merging of the assemblies produces a complete sequence that can be evaluated for gene content, single nucleotide polymorphisms (SNPs) and structural variation (SV; indels, inversion, translocations) both by web-based searches of external databases to identify known allelic variation and by direct examination of the sequence to identify new polymorphisms. A Disk Sort Alignment algorithm allows the data sets which are far too large for in-memory processing to be evaluated and clustered for assembly by SeqMan N-Gen (SM N-Gen), our desktop assembly engine. Using a prototype DSA-SM N-Gen pipeline, we have processed the entire 7.4x 454 data set from the James Watson genome to a layout file in 31 hours using DSA and have assembled three chromosomes: 8; 21; and X; using SM N-Gen. Assembly times varied from 1 hour for Chromosome 21 to 10.6 hours for an average- sized chromosome, such as Chromosome 8. Together, these results demonstrate the feasibility of constructing a DESKAPP pipeline for WHGR. The Phase II Aims are designed to build upon this foundation and produce a seamless pipeline for the desktop assembly and analysis of a human genome in a matter of days. PUBLIC HEALTH RELEVANCE: Next-gen sequencing technologies have started a new revolution throughout biology by providing DNA sequence data in unprecedented quantities at continually decreasing costs. This data will be invaluable in the emerging era of personalized medicine and in exploring the immense diversity of life. The goal of this project is to develop desktop computer software that will enable research laboratories and clinics of any size to realize the promise of these new technologies.