Next-Generation Sequencing: From Basic Research to Diagnostics

Abstract

Background: For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). Content: This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. Summary: In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.

Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

“…The first part of this study was mainly aimed at validation of the enrichment protocol (proof of concept, patients 1-10), whereas the second part of this study consisted of a blind screening of 12 prescreened mutation-negative patients with LCA (patients [11][12][13][14][15][16][17][18][19][20][21][22]. enrichment of LcA disease genes qPCR was used to target all exons from 16 LCA disease genes.…”

“…For the second lane, libraries were prepared from a 200-300-bp size-selected fraction, and library quantification was performed using qPCR following manufacturer's protocols (lane 2, patients [11][12][13][14][15][16][17][18][19][20][21][22]. In total, 120 µl of a pool of the 12 normalized libraries (concentration of 10 pmolar) was subjected to paired-end sequencing of 2 × 45 cycles.…”

“…This study included 17 patients in which previous Sanger sequencing of six genes and/or LCA chip analysis did not identify causal mutations (lane 1: patients 1-3, 7, and 8; lane 2: patients [11][12][13][14][15][16][17][18][19][20][21][22]. Given the exclusion of 3 of 107 known variants by the in 12.7% of the Yoruba population (1000Genomes Project, pilot_1_YRI_low_coverage_panel) points to a polymorphism rather than a mutation (rs61748445).…”

“…18 However, a major challenge remains to enrich regions of interest, such as exons, from the patient's genome. Although hybridization-based DNA capture is often used, this approach has several limitations, including a high cost, suboptimal capturing efficiency at repetitive regions, interference from homologous sequences, a large variation in coverage, and lack of flexibility if new regions need to be captured.…”

Advances in genomics for flatfish aquaculture

“…Thereafter, the clusters of DNA templates are sequenced by synthesis or ligation in a phased approach. There are three main SGS platforms (reviewed by Glenn 2011;Metzker 2010;Voelkerding et al 2009): (a) the 454 platform (Roche Applied Science), based on emulsion PCR followed by pyrosequencing reactions to produce approximately 0.4-0.5 gigabases (Gb) per run rendering sequences of 300-400 base pairs (bp) mean length (although a new chemistry is ongoing to reach 600-700 bp mean length) (e.g. 454 FLX Titanium sequencer); (b) the SolexaIllumina Ò platform (Illumina, Inc.), which uses bridge PCR for DNA amplification and dye-labelled terminators in a polymerase-mediated reaction for sequencing, and produces 200-300 Gb/run with a sequence mean length of 100 bp (e.g.…”