Anthony Nolan Research Institute, Royal Free Hospital, London, UK.
Immunogenetics. 2010 Apr;62(4):253-60. doi: 10.1007/s00251-010-0432-5. Epub 2010 Mar 4.
Sequence-based typing (SBT) is a powerful method of genotyping in highly polymorphic gene systems. In standard SBT methods, both strands of a double-stranded template amplicon are sequenced in separate reactions in order to achieve high quality data across the region of interest. The amount of informative data that is obtained from the second strand sequence is often low, whilst the impact of performing second strand sequencing on costs and throughput are significant. Here we present short template amplicon and multiplex megaprimer-enabled relay (STAMMER) sequencing, a novel simultaneous sequence-based typing methodology that allows the detection of any practical amount of useful sequence from a plurality of distinct polymerase chain reaction products in a single sequencing reaction. In addition to simultaneous bidirectional sequencing, we show how the STAMMER approach can be used to simultaneously sequence a number of regions of interest that are not physically linked within the range of a single sequencing reaction. The efficiencies of this method could impact significantly on the output of SBT laboratories.
基于序列的分型（SBT）是一种在高度多态性基因系统中进行基因分型的强大方法。在标准的 SBT 方法中，双链模板扩增子的两条链都在单独的反应中进行测序，以在感兴趣的区域获得高质量的数据。从第二条链序列获得的信息量通常较低，而进行第二条链测序对成本和通量的影响是显著的。在这里，我们提出了短模板扩增子和多重大片段引物辅助接力（STAMMER）测序，这是一种新颖的同时基于序列的分型方法，允许在单个测序反应中从多个不同的聚合酶链反应产物中检测到任何实际数量的有用序列。除了同时双向测序外，我们还展示了如何使用 STAMMER 方法同时对在单个测序反应范围内不物理连接的多个感兴趣区域进行测序。这种方法的效率可能会对 SBT 实验室的产量产生重大影响。
