A platform for massive parallel sequencing of longPCR amplicons

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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.