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Next Generation Sequencing (NGS)

Next Generation Sequencing (NGS) is a technology that enables the rapid and affordable sequencing of large amounts of genetic material (DNA or RNA). Compared to more traditional methods, such as the Sanger Sequencing developed in 1977, NGS protocols are often also referred to as 2nd or 3rd generation sequencing. The ability to simultaneously sequence millions of DNA fragments in parallel has greatly contributed to its advantage over earlier classical sequencing platforms. Furthermore, the capability to quickly sequence very large sample numbers not only reduced the time and cost but also drastically increased affordability of amassing an ever-growing body of available sequence data. Combined with advancements made in bioinformatics tools and digital analysis, this also increased the overall sensitivity and accuracy. NGS has thus become a powerful tool for scientists to identify previously undetectable variations, and to address previously intractable problems in basic and clinical biology.

NGS technologies involve three basic steps: library construction, sequencing process, and data analysis. During library construction, DNA is fragmented through various established methods into many short segments. These are then further processed for library construction in various ways. During this process the DNA segments are modified for identification of a specific target sequence. Adaptors are typically added, allowing the sequence to bind to a complementary sequence. Through an amplification process the DNA segments are enriched, and the library is thus constructed applying established procedures. Sequencing is carried out on one of a number of established contemporary NGS sequencer platforms.  The library data are generally uploaded into a sequencer matrix. The generated sequence information is analyzed using advanced bioinformatic tools, which routinely involves base calling, read alignment, variant identification, and variant annotation. During this process the sequence information is frequently compared to a reference genome for identification of the presence or the absence of variants as well as their relevant biological and clinical significance.

The capacity of NGS to sequence either whole genomes, whole exomes, various transcriptome formats, or panels of targeted sequences (e.g. detection of copy number variations, single nucleotide polymorphisms, mutations ranging from single point mutations to lengthy aberrations, and large scale structural variations) has greatly contributed to its expansive application in basic research, prenatal and postnatal genetic testing, and nearly all facets of clinical medicine, particularly in cancer diagnosis, prognosis, and development of novel therapeutics.



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