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Sequencing

In next generation sequencing workflows, nucleic acid is extracted from a sample and fragmented, arranged into platform-specific library constructs, amplified and sequenced. Regardless of the sample type or the platform used, every step throughout this workflow is critical for successful results. 

Promega products to support NGS workflows include nucleic acid extraction and quantification systems, and Pronex® NGS Quantitation and Size Selective DNA Purification Systems for library preparation prior to sequencing.

Sequencing and NGS Basics

From basic science to translational research, next-generation sequencing (NGS, also known as massively parallel sequencing) has opened up new avenues of inquiry in genomics, oncology and ecology. The availability of NGS technology has made sequencing a routine and viable option for diagnostic, forensic and epidemiological investigations and has enabled advances in many genomic analysis applications.

In Sanger Sequencing (first-generation sequencing) DNA fragments are sequenced by the incorporation of chain terminating nucleotides, which are then separated by electrophoresis and detected by a fluorescent signal. In NGS, millions of DNA fragments are sequenced in parallel and nucleotides are detected as they are added to the DNA strand. After DNA extraction and fragmentation, clusters of each DNA template are amplified by PCR, and attached to a solid surface. They are then interrogated with nucleotides and imaged/measured as the DNA is sequenced. 

There are several NGS technologies available: Illumina sequencers use reversible terminator dye-labeled nucleotides to interrogate the captured DNA. Once each base is read, the terminator and dye are removed by cleavage and washing, creating a normal nucleotide. The strand is once again extensible and the process is repeated to continue sequencing along the strand. Instead of using dye-labeled nucleotides, the Ion Torrent sequencer measures the release of hydrogen ions upon base incorporation, and the 454 system measures luminescence upon nucleotide incorporation. These sequencers can process large numbers of samples in parallel, increasing speed and throughput, and making sequencing of whole genomes in short timeframes both achievable and affordable.