Adapting Phred/Phrap/Consed for NextGen Sequencing

描述（由申请人提供）：使Phred/Phrap/Consed适应下一代测序DNA测序的新方法能够以传统技术的一小部分成本产生更多的数据，因此DNA测序现在在生物医学研究中的应用比以往任何时候都多。然而，分析这些新技术产出的软件可以得到显著改进。本提案旨在为这些“下一代”测序仪升级广泛使用的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，该程序比Illumina软件的读取量多80%-90%，碱基调用错误减少50%，从而显着降低测序成本，并允许更自信地检测序列变异。我们将进一步改进性能，并研究改变Illumina实验方案是否能进一步提高产量。我们还将校正其他下一代测序仪的碱基调用者的误差概率。我们将使consed（可视化、整理和分析工具）能够灵活地处理多达数十亿个reads的汇编、大型参考序列和高覆盖深度；利用概率模型检测结构变异并确定snp；直接读取下一代数据常用的汇编程序的输出；并对错误装配和共识基础进行批量修正。我们将进一步改进cross_match（灵活的序列比对程序，是phred/phrap/consed的一部分）和我们新的超快速比对器，用于将大量基因组或RNA-Seq读取到参考基因组。这两个程序的速度和功能都将得到增强，包括处理配对读取和以更紧凑的文件格式输出对齐的能力。我们将创造一个生物信息学环境，即使是小实验室也能管理下一代测序仪的大量数据。这将包括紧凑文件格式的实现，数据存储的处方，各种应用程序中可用文件的生成，以及Illumina和454数据处理的管道。

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.