使用454 GS FLX系统进行下一代HLA测序。
Next-generation HLA sequencing using the 454 GS FLX system.
作者信息
Trachtenberg Elizabeth A, Holcomb Cherie L
机构信息
Children's Hospital & Research Center, Oakland, CA, USA.
出版信息
Methods Mol Biol. 2013;1034:197-219. doi: 10.1007/978-1-62703-493-7_10.
Next-generation sequencing (NGS) of HLA class I and II loci (HLA-A, HLA-B, HLA-C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPB1) is described here in detail using the 454 Life Sciences GS FLX System and Titanium chemistry. An overview of the protocol with our experience on sequence performance efficiencies, read depth and ambiguity analyses using the GS FLX System are also presented. A total of 14 HLA primer pairs with multiplex identifiers (MIDs) are used in clonal, amplicon-based pyrosequencing of up to 44 samples per plate using the GS FLX. Genotype assignment and ambiguity reduction -analysis is performed using Conexio Assign ATF 454 software. Clonal NGS gives a significant reduction in genotyping ambiguity during analysis of the highly complex HLA system.
本文详细介绍了使用454生命科学公司的GS FLX系统和钛化学技术对HLA I类和II类基因座（HLA-A、HLA-B、HLA-C、DRB1、DRB3、DRB4、DRB5、DQA1、DQB1、DPB1）进行下一代测序（NGS）的方法。文中还概述了该方案以及我们在使用GS FLX系统进行序列性能效率、读取深度和歧义分析方面的经验。总共14对带有多重标识符（MID）的HLA引物对用于基于克隆扩增子的焦磷酸测序，使用GS FLX系统时每板最多可对44个样本进行测序。使用Conexio Assign ATF 454软件进行基因型分配和歧义减少分析。在高度复杂的HLA系统分析过程中，克隆式NGS显著降低了基因分型中的歧义。
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