29.11.2016 NGS: Next Generation Sequencing Background DNA sequencing: Process which establishes the order of the nucleotides (building blocks) of a DNA molecule – adenine, guanine, cytosine and thymine. In 1977, F. Sanger described a protocol using “chain terminating inhibitors” and his method, the Sanger method, initially using radioactive labelled nucleotides and slab gels, amplified the “DNA revolution” started by Watson and Crick in 1953. In 1995-1997, the method evolved to allow higher throughput. Development of capillary systems, fluorochrome labelled nucleotides and automated reading platform made genome projects and micro population screening possible. In 2003, after 13 years and a cost exceeding $2.5 billion, the Human Genome Project was declared complete, mainly produced using this methodology. In 2005, Next Generation Sequencing (NGS) became a reality with a complete revolution of the method and the amount of data a single run would produce. Principle Sanger sequencing is considered the gold standard method, however, its limitation is that it requires pure samples and cannot be used to analyse complex matrices in a single simple procedure, i.e. mixed sequences originating from adulterated samples, microbial community etc. The method can only read one DNA sequence type at a time. Therefore a mixed sample of meats would require several additional stages of analysis to identify all its components (multi-PCR, cloning, purification etc.). The principle of NGS is based on massive parallel sequencing, which generates thousands of megabases of sequence on a single run. Next-generation techniques are based on a “sequencing by synthesis” principle, where nucleotide incorporates into a strand of DNA and each addition creates a detectable reaction. The reactions can take form of a fluorescent molecule or a pH change but the detection of these on an extreme scale is where NGS out-performed the Sanger by thousands of times. The method is therefore designed to cope with numerous different targets at once and provide the perfect answer for the type of analysis like adulteration, authentication test. Using NGS removes the need for fastidious, highly biased and time consuming steps required for Sanger method to decipher informations originating form complex samples. Sanger sequencing (from Jay Shendure amp; Hanlee Ji, Nature Biotechnology 26, 1135 – 1145 (2008)) NGS applications Since the meat Horsegate and the clear focus initiated by BRC Global Standard for Food Safety Issue 7 on traceability/authentication, the Food industry and Retailers in general have been looking at new methodologies and techniques. (from Jay Shendure amp; Hanlee Ji, Nature Biotechnology 26, 1135 – 1145 (2008)) NGS is one of these techniques and is particularly applicable to: Authentication of complex samples: Fruit/Crops : possible origin statement Plant cultivar Tackling adulteration with a blind approach: until recently we could only detect the presence or absence of a specific target. NGS offers the possibility of a blind approach, finding all the components without knowledge of what they are. Analysis of microbial communities (Many organisms are not detected by conventional culturing methods) Identification and characterisation of microbes: revolution in multi-locus sequence typing.