Next-generation sequencing

Next-generation sequencing
Name	Next-generation sequencing
Caption	High-throughput sequencing instrument
Invented	2005–2010
Inventors	Multiple groups
Application	Genomics, transcriptomics, metagenomics

Next-generation sequencing

Next-generation sequencing revolutionized molecular biology by enabling massively parallel DNA and RNA sequencing. The technology displaced earlier Sanger sequencing in many settings and catalyzed projects such as the Human Genome Project follow-on initiatives, large-scale efforts by the National Institutes of Health, and private endeavors at companies like Illumina, Inc. and Ion Torrent. It accelerated research across institutions including Broad Institute, Wellcome Sanger Institute, European Bioinformatics Institute, and clinical programs at Mayo Clinic and Massachusetts General Hospital.

History and development

Early concepts trace to advances in enzymology and instrumentation pioneered by laboratories associated with Frederick Sanger and institutions like Cambridge University. Key milestones include the introduction of reversible terminator chemistry by Illumina, Inc., semiconductor sequencing by Ion Torrent (a division of Life Technologies), and single-molecule real-time methods developed by Pacific Biosciences and nanopore approaches commercialized by Oxford Nanopore Technologies. Large consortia such as the 1000 Genomes Project and efforts coordinated by the National Human Genome Research Institute shaped standards, while private funding from entities linked to Craig Venter and foundations like the Bill & Melinda Gates Foundation influenced application-driven growth.

Technologies and platforms

Platforms differ by chemistry and read architecture. Sequencing-by-synthesis approaches from Illumina, Inc. use reversible terminators and patterned flowcells. Semiconductor sequencing from Ion Torrent detects pH changes, whereas single-molecule real-time sequencing from Pacific Biosciences leverages zero-mode waveguides. Nanopore sequencing by Oxford Nanopore Technologies measures ionic current through protein pores. Other contributors and vendors include Roche Diagnostics (454 pyrosequencing legacy), Thermo Fisher Scientific (Ion Torrent lineage), and academic groups at Harvard University and Stanford University that developed early high-throughput platforms.

Methodology and workflows

Typical workflows start with sample collection at hospitals like Johns Hopkins Hospital or biobanks such as the UK Biobank, followed by nucleic acid extraction protocols standardized by the World Health Organization or regulatory frameworks from the Food and Drug Administration. Library preparation methods incorporate fragmentation, adapter ligation, and amplification steps influenced by kits from New England Biolabs and protocols from research centers at Cold Spring Harbor Laboratory. Cluster generation or template prep differs by platform; sequencing runs occur on instruments maintained by core facilities at universities like University of California, Berkeley or corporate service labs such as BGI Group. Quality control uses metrics rooted in standards set by organizations including the International Organization for Standardization.

Applications

High-throughput sequencing supports clinical genomics in oncology clinics at Memorial Sloan Kettering Cancer Center and rare disease diagnosis through programs like the Undiagnosed Diseases Network. It powers population genomics projects led by deCODE genetics and epidemiological surveillance in public health agencies such as the Centers for Disease Control and Prevention. Environmental and metagenomic studies are conducted by teams at Scripps Institution of Oceanography and institutions participating in the Earth Microbiome Project. Agricultural genomics efforts involve corporations like Monsanto/Bayer and academic centers such as Iowa State University. Transcriptome profiling informs research at Stanford University School of Medicine and vaccine development at organizations including Moderna, Inc..

Data analysis and bioinformatics

Sequencing data processing pipelines use alignment tools and variant callers developed in groups at Broad Institute and universities like University of Cambridge; common software originates from projects associated with Genome Analysis Toolkit and community efforts coordinated at GitHub. Reference datasets include assemblies from Genome Reference Consortium and annotations curated by Ensembl and RefSeq at National Center for Biotechnology Information. Compute infrastructure relies on cloud services from Amazon Web Services, Google Cloud Platform, and high-performance clusters at national labs such as Lawrence Berkeley National Laboratory. Training and reproducibility are supported by courses at European Molecular Biology Laboratory and standards promoted by the Global Alliance for Genomics and Health.

Limitations, challenges, and quality control

Challenges include read-length and error profiles characterized in technical reports by vendors like Illumina, Inc. and Pacific Biosciences, batch effects noted by researchers at Johns Hopkins University, and contamination incidents investigated by teams at Wellcome Sanger Institute. Regulatory oversight and validation pathways involve submissions to the Food and Drug Administration and guidelines from the College of American Pathologists. Cost, throughput, and supply-chain constraints were highlighted during public health crises addressed by World Health Organization coordination. Quality control employs metrics established by consortia such as the 1000 Genomes Project and proficiency testing from organizations like the Clinical Laboratory Improvement Amendments program.

Ethical, legal, and social implications

Use of sequencing in clinical and population settings intersects with policies from the Health Insurance Portability and Accountability Act and debates in forums organized by the National Academy of Medicine. Issues include privacy concerns raised in litigation and scholarship involving entities like American Civil Liberties Union and policy analyses by the European Commission. Equity and access initiatives are championed by global programs funded by the Bill & Melinda Gates Foundation and implementation projects at Médecins Sans Frontières. Intellectual property disputes have involved companies such as Illumina, Inc. and Oxford Nanopore Technologies, while ethical frameworks are discussed in reports from the Nuffield Council on Bioethics and working groups at the World Health Organization.

Category:Genomics