Integrated Bioinformatics Analysis Suite for deep, targeted amplicon sequencing

描述(由申请人提供)：这项提案的目的是开发一种生物信息学管道，或集成生物信息学分析套件(IBAS)，专门用于在新的Ion Torrent个人基因组机器(PGM)上进行超深度、有针对性的扩增子测序。这将提高变异检测的准确性，并使分析不同类型癌症的基因变异成为可能。PGM中使用的技术基于半导体技术的非光学检测，因此具有足够的独特性，能够在使用其他技术进行初始测序时用作验证测试的正交平台。与其他下一代测序方法相比，PGM提供了一个显著的优势，使样品准备到数据生成的过程可以在一天内完成。然而，它受到技术的内在限制，主要是32端的错误。此外，目前可用的SNP呼叫者不是特别适合高吞吐量、超 深度扩增子测序，几乎没有算法可用于检测其他类型的基因组变异。拟议的管道旨在解决这些问题，并满足对生物信息学的迫切需求，该生物信息学针对PGM所采用的面板设计的扩增子测序技术进行了优化。为了实现拟议的目标，1)将构建一个IBAS，它包括不同的方法来读取预处理、与参考序列比对和错误建模，以便能够检测单核苷酸多态(SNPs)、插入/缺失(INDELs)、拷贝数变化(CNV)和其他结构变化；以及2)将对变异源进行综合评估，并针对扩增子测序进行优化。然后，将通过处理来自PGM的数据来验证优化的流水线，以对临床甲状腺细针抽吸活检中检测到的突变进行正交验证。成功的结果将使Asuragen能够为临床研究和诊断提供高通量超深度测序服务。 公共卫生相关性：我们的目标是开发一种生物信息学应用程序，专门用于最近推出的离子洪流个人基因组机(PGM)所采用的技术，该应用程序将提高检测不同类型癌症的基因变异的准确性。PGM是一种紧凑、台式的下一代测序仪器，能够以比现有系统低得多的成本快速对整个基因组进行测序。Asuragen将使用该应用程序为临床研究和诊断提供高通量测序服务。

DESCRIPTION (provided by applicant): The aim of this proposal is to develop a bioinformatics pipeline, or Integrated Bioinformatics Analysis Suite (IBAS), specific for ultra-deep, targeted amplicon sequencing on the new Ion Torrent Personal Genome Machine (PGM). This will improve the accuracy of variation detection and enable analysis of genetic variations in different types of cancer. The technology used in the PGM is based on non-optical detection with semiconductor technology and so is sufficiently unique to enable its use as an orthogonal platform for confirmatory testing when other technologies are used for initial sequencing. The PGM offers a significant advantage over other methods of next generation sequencing by enabling the process of sample preparation to data generation to be completed in one day. However, it suffers from the inherent limitations of the technology, mainly errors at the 32 end. Additionally, currently available SNP callers are not particularly suited to high-throughput, ultra deep amplicon sequencing and there are few algorithms available for detecting other types of genomic variations. The proposed pipeline aims to address these issues and fill the urgent need for bioinformatics optimized for the panel-designed amplicon sequencing technology employed in the PGM. To achieve the proposed aims, 1) an IBAS that incorporates different approaches to read preprocessing, alignment to reference sequences, and error modeling to enable detection of single nucleotide polymorphisms (SNPS), insertion/deletions (indels), copy number variation (CNV) and other structural variation will be constructed; and 2) a comprehensive evaluation of the sources of variation will be performed, and the IBAS optimized for amplicon sequencing. The optimized pipeline will then be validated by processing data from the PGM for orthogonal validation of mutations detected in clinical thyroid fine needle aspiration biopsies. Successful outcome will enable Asuragen to provide a high-throughput ultra-deep sequencing service for clinical research and diagnostics. PUBLIC HEALTH RELEVANCE: We aim to develop a bioinformatics application specific for the technology employed in the recently launched Ion Torrent Personal Genome Machine (PGM) that will improve the accuracy of detecting genetic variations in different types of cancer. The PGM is a compact, bench-top next generation sequencing instrument that enables rapid sequencing of an entire genome and at a much lower cost than established systems. The application will be used by Asuragen to offer a high-throughput sequencing service for clinical research and diagnostics.