Adapting Phred/Phrap/Consed for NextGen Sequencing

描述(申请人提供)：使Phred/Phrap/Consed适应下一代测序DNA测序的新方法允许以传统技术的一小部分成本产生更多的数据，因此DNA测序现在比以往任何时候都更多地用于生物医学研究。然而，分析这些新技术的输出的软件可能会得到显着改进。这项提议是为了升级这些“下一代”测序仪广泛使用的Phred/Phrap/Consed封装。我们为Illumina测序仪开发了一种新的碱基调用和图像分析程序Next_Phred，它的读数比Illumina软件多80%-90%，碱基调用错误减少50%，从而显著降低测序成本，并允许更可靠地检测序列变异。我们将进一步提高性能，并调查对Illumina实验协议的更改是否可以进一步提高产量。我们还将校准其他下一代定序器的基调用方的错误概率。我们将使COSTED(可视化、整理和分析工具)能够灵活地处理高达数十亿次读取、大型参考序列和高覆盖深度的程序集；使用概率模型检测结构变异并确定SNP；直接读取与下一代数据常用的汇编器的输出；以及对错误的程序集和共识基础执行批量更正。我们将进一步改进CROSS_MATCH(灵活的序列比对程序，它是Phred/Phrap/COSED的一部分)和我们新的超快速比对程序，用于将大量基因组或RNA-Seq读数映射到参考基因组。这两个程序都将得到速度和功能的增强，包括处理成对读取和以更紧凑的文件格式输出比对的能力。我们将创造一个生物信息学环境，让即使是小型实验室也能管理来自下一代测序仪的海量数据。这将包括实施紧凑的文件格式，规定数据存储，生成可用于各种应用程序的文件，以及Illumina和454数据处理的管道。 与公共卫生相关：新的DNA测序技术正在极大地增加可用于破译人类疾病遗传基础的数据量。目前还缺乏能够充分利用这些数据的软件。我们的软件通常用于老式测序机，我们将对其进行改进，以迎接这一挑战并显著降低测序成本。

DESCRIPTION (provided by applicant): Adapting Phred/Phrap/Consed to Next-Generation Sequencing New methods for DNA sequencing are allowing the production of much more data at a fraction of the cost of traditional technologies, such that DNA sequencing is now being used more than ever before in biomedical research. However software to analyze the output from these new technologies could be significantly improved. This proposal is to upgrade the widely used phred/phrap/consed package for these "next-generation" sequencers. We have developed a new base-calling and image analysis program, next_phred, for the Illumina sequencer which gives 80%-90% more reads than the Illumina software and 50% fewer base- calling errors, thus significantly reducing sequencing costs and allowing more confident detection of sequence variants. We will make further performance improvements and investigate whether changes to the Illumina experimental protocol can increase yield still further. We will also calibrate the error probabilities for the base-callers of other next-generation sequencers. We will enable consed (the visualization, finishing, and analysis tool) to nimbly handle assemblies of up to several billion reads, a large reference sequence, and high depth of coverage; to detect structural variants and determine SNPs using a probabilistic model; to directly read the output of assemblers commonly used with next-generation data; and to perform batch correction of erroneous assemblies and consensus bases. We will further improve cross_match (the flexible sequence alignment program which is part of phred/phrap/consed) and our new ultrafast aligner phaster for mapping large numbers of genomic or RNA-Seq reads to a reference genome. Both programs will be given speed and functionality enhancements, including the capability to handle paired reads and to output alignments in a more compact file format. We will create a bioinformatics environment allowing even small labs to manage the massive amounts of data from next-generation sequencers. This will include the implementation of compact file formats, prescriptions for data storage, generation of files usable in a variety of applications, and pipelines for Illumina and 454 data processing. PUBLIC HEALTH RELEVANCE: New DNA sequencing technologies are vastly increasing the amount of data available to decipher the genetic basis of human disease. Software able to fully exploit this data is currently lacking. Our software, commonly used for older types of sequencing machines, will be improved to meet this challenge and to significantly lower sequencing costs.