Summary Biological Techniques

Biological TechniquesTable of contents  Introduction  Fundamentals of NGS  Potential uses of NGS a. Microbiology b. oncology  NGS Methods Genomics a. Whole genome sequencing b. Exome sequencing c. De novo sequencing Transcriptomics a. Total RNA and mRNA sequencing b. Targeted RNA sequencing Epigenomics a. Methylation sequencing  Limitations  References Next generation sequencing Next generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology which has revolutionised genomic research. Using NGS an entire human genome can be sequenced within a single day. In contrast, the previous Sanger sequencing technology, used to decipher the human genome, required over a decade to deliver the final draft. Although in genome research NGS has mostly superseded conventional Sanger sequencing, it has not yet translated into routine clinical practice. There are a number of different NGS platforms using different sequencing technologies, all NGS platforms perform sequencing of millions of small fragments of DNA in parallel. Bioinformatics analyses are used to piece together these fragments by mapping the individual reads to the human reference genome. Each of the three billion bases in the human genome is sequenced multiple times, providing high depth to deliver accurate data and an insight into unexpected DNA variation. NGS can be used to sequence entire genomes or constrained to specific areas of interest, including all 22 000 coding genes (a whole exome) or small numbers of individual genes. Fundamentals of NGS Platforms NGS platforms share a common technological feature massively parallel sequencing of clonally amplified or single DNA molecules that are spatially separated in a flow cell. This design is a paradigm shift from that of Sanger sequencing, which is based on the electrophoretic separation of chaintermination products produced in individual sequencing reactions. In NGS, sequencing is performed by repeated cycles of polymerase-mediated nucleotide extensions or, in one format, by iterative cycles of oligonucleotide ligation. As a massively parallel process, NGS generates hundreds of mega bases to giga bases of nucleotide sequence output in a single instrument run, depending on the platform. These platforms are reviewed next. POTENTIAL USES OF NGS IN CLINICAL PRACTICE