A platform for massive parallel sequencing of longPCR amplicons

新一代测序技术提供了一种前所未有的基因和基因组测序方法，成本只有以前的一小部分，覆盖面也非常大。各种平台提供不同的技术。454焦磷酸测序，也被称为大规模平行测序，其优点是在微滴定板上的100多万个单独的反应室中提供相对较长的序列读数(~450个核苷酸)；正在开发捕获更长的读数的技术。当混合来自不同来源的模板时，需要将序列与其来源链接。有两种可能的方法，包括(I)在单个微滴定板或板的单个衬垫部分(最多16个)上处理单个样品，或(Ii)用独特的样品特异性标记对模板进行化学标记。长链DNA(高达20,000个核苷酸)通过特殊的聚合酶链式反应进行常规扩增，以用于各种目的，由各种分子工具的用户进行。通过使用传统方法对这些长扩增片段的末端进行测序，并依靠生物信息学工具准确解读数据，我们提出了一种方法，允许对数百个长扩增片段进行汇集、碎片化、大规模并行测序、准确重组和识别，从而将现有成本降低数量级。这项技术将允许对长PCR进行常规的多重测序，在这种情况下，以前只能对短片段进行测序，或者需要昂贵的特定样本标记和/或克隆。我们将通过从寄生蠕虫产生长的PCR扩增来测试该方法，对这些寄生虫：(I)我们有各种各样的样本和丰富的处理经验，(Ii)人们对此有广泛的兴趣和需求，包括诊断学、生物多样性研究和寄生虫学进化。模拟研究将与实际数据结合使用，以开发、完善和测试生物信息学管道，以便更广泛地应用。这种方法和相关的开放获取计算机应用程序将可转移到任何生物系统，在该系统中，无论DNA的来源如何，都会对不同的长PCR片段进行测序。

New generation sequencing techniques offer an unprecedented means of sequencing genes and genomes at a fraction of previous costs and at a phenomenal density of coverage. A variety of platforms offer different techniques. 454 pyrosequencing, also known as massive parallel sequencing, has the advantage of providing relatively long sequence reads (~450 nucleotides) in over 1 million individual reaction chambers on a pico-titre plate; developments are under way to capture even longer reads. When mixing templates from different sources there is a need to link sequences with their source. Two ways are possible and include (i) processing individual samples on single pico-titre plates or individual gasketed sections of a plate (up to 16), or (ii) chemically-tagging templates with unique sample-specific markers. Long lengths of DNA (up to 20,000 nucleotides) are routinely amplified with specialised polymerase chain reactions for a diversity of purposes by a wide variety of users of molecular tools. By sequencing the ends of these long amplicons using traditional methods, and by relying on bioinformatic tools to accurately unscramble the data, we propose a method that allows hundreds of long amplicons to be pooled, fragmented, massively parallel sequenced, accurately reassembled and identified, thus reducing existing costs by orders of magnitude. The technique will allow routine multiplex sequencing of longPCRs where only short fragments could be sequenced previously, or where expensive sample-specific tagging and/or cloning was required. We will test the methodology by generating longPCR amplicons from parasitic helminths, for which: (i) we have a wide diversity of samples available and considerable experience of handling, (ii) there is wide interest and need, including diagnostics, biodiversity studies and evolutionary parasitology. Simulation studies will be used in conjunction with real data to develop, refine and test the bioinformatics pipeline for wider application. The methodology and associated open access computer applications will be transferable to any biological system where diverse longPCR fragments are sequenced regardless of the origin of the DNA.