title: Understanding the Basics of NGS: From Mechanism to Variant Calling.
abstract: Identifying disease-causing mutations in DNA has long been the goal of genetic medicine. In the last decade, the toolkit for discovering DNA variants has undergone rapid evolution: mutations that were historically discovered by analog approaches like Sanger sequencing and multiplex ligation-dependent probe amplification ("MLPA") can now be decoded from a digital signal with next-generation sequencing ("NGS"). Given the explosive growth of NGS-based tests in the clinic, it is of the utmost importance that medical practitioners have a fundamental understanding of the newest NGS methodologies. To that end, here we provide a very basic overview of how NGS works, with particular emphasis on the close resemblance between the underlying chemistry of Sanger sequencing and NGS. Using a pair of simple analogies, we develop an intuitive framework for understanding how high-confidence detection of single-nucleotide polymorphisms, indels, and large deletions/duplications is possible with NGS alone.
authors.affiliation: Counsyl Inc., 180 Kimball Way, South San Francisco, CA 94080 USA.
authors.forename: Dale
authors.initials: D
authors.lastname: Muzzey
authors.affiliation: Counsyl Inc., 180 Kimball Way, South San Francisco, CA 94080 USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Evans
authors.affiliation: Counsyl Inc., 180 Kimball Way, South San Francisco, CA 94080 USA ; Joan H Marks Graduate Program in Human Genetics, Sarah Lawrence College, Bronxville, NY USA.
authors.forename: Caroline
authors.initials: C
authors.lastname: Lieber
keywords: Del/dup calling; Next-generation sequencing (NGS); Read depth; SNP/indel calling; Variant calling