Kothiyal Prachi, Cox Stephanie, Ebert Jonathan, Aronow Bruce J, Greinwald John H, Rehm Heidi L
Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.
Curr Protoc Hum Genet. 2009 Apr;Chapter 7:Unit 7.17. doi: 10.1002/0471142905.hg0717s61.
This unit provides an overview of oligo hybridization-based resequencing and a wide range of considerations for implementing the technology and analyzing the resulting data. The specific technology discussed is the Affymetrix GeneChip CustomSeq Resequencing Array platform. Concepts related to array design, experimental protocols, and base-calling using existing algorithms are presented. Details that should be evaluated during development of sequence tiling, target amplification, and PCR protocols are addressed. An overview of the Affymetrix GeneChip Sequence Analysis Software (GSEQ) is provided, along with factors that influence base-calling coverage and accuracy. Also outlined are performance measures that can be used to characterize base-calling with resequencing arrays, as well as factors known to affect their performance. Limitations associated with detection of insertions and deletions (indels) are discussed, with empirical data from our experiments used to outline possible approaches to indel detection. Critical topics in the design, implementation, and analysis of targeted sequencing arrays not previously discussed in detail are highlighted.
本单元概述了基于寡核苷酸杂交的重测序技术，并阐述了实施该技术及分析所得数据时需要考虑的诸多因素。所讨论的具体技术是Affymetrix GeneChip CustomSeq重测序阵列平台。文中介绍了与阵列设计、实验方案以及使用现有算法进行碱基识别相关的概念。探讨了在序列拼接、目标扩增和PCR方案开发过程中应评估的细节。概述了Affymetrix GeneChip序列分析软件（GSEQ），以及影响碱基识别覆盖率和准确性的因素。还概述了可用于表征重测序阵列碱基识别的性能指标，以及已知会影响其性能的因素。讨论了与插入和缺失（indel）检测相关的局限性，并利用我们实验中的经验数据概述了检测indel的可能方法。强调了在靶向测序阵列的设计、实施和分析中以前未详细讨论的关键主题。
