Massively parallel sequencing

Sanger sequencing revolutionised sequencing technology; however, methods now exist that allow for accurate results in faster and more cost-efficient ways. Massively parallel sequencing, also known as next-generation sequencing, can sequence millions of fragments of DNA at the same time, something that is not possible using Sanger. This breakthrough has paved the way for the application of DNA sequencing in routine care for a variety of clinical scenarios.

Massively parallel sequencing is used clinically for whole genome sequencing (WGS), whole exome sequencing (WES), gene panel testing, and increasingly for single gene testing.

These genomic technologies have transformed the fields of oncology, rare disease, infectious disease and prenatal diagnostics, and are increasingly becoming an integral part of mainstream clinical practice across all healthcare specialties.

Massively parallel sequencing is also extensively used for research, enabling an era of gene discovery and diagnosis of rare monogenic disorders, as well as the identification and diagnosis of genetic factors contributing to common complex disease.

Massively parallel sequencing also underpinned the world-leading 100,000 Genomes Project, a pioneering study in which WGS was first offered to NHS rare disease and cancer patients.

Advantages

• Multiple genes can be tested in a single assay, reducing the diagnostic odyssey associated with offering patients successive single gene tests.
• Samples for different patients can be tested together in batches, improving cost effectiveness.
• Massively parallel sequencing has a high sensitivity for single nucleotide changes and small deletions or insertions (indels).
• In some cases, copy number variation and even structural variants (WGS only) can also be detected.

Clinical situations in which massively parallel sequencing is particularly useful include those in which many possible genes and variant types could be causing a patient’s features.

Limitations

• Where lots of genes are tested, careful filtering and interpretation of variants is required, as most are unlikely to be causal.
• Incidental findings may be a concern where many genes are tested.
• Large amounts of data are generated, requiring sufficient storage.
• Some regions may not have enough sequencing reads. In any such ‘gaps in coverage’, variants might be missed.
• Copy number variants may not be detected as reliably as single nucleotide variants or small indels.
• Structural variants may be detected by massively parallel sequencing, but these would not usually be detected by targeted panel sequencing.
• Currently, methylation changes are not detected, and repeat expansion disorders may be detected but are usually tested for separately.

For a summary table comparing the advantages and disadvantages of the different approaches to gene sequencing (gene panel sequencing, WES and WGS), see Different approaches to gene sequencing.

• Massively parallel sequencing underpins the clinical applications for WGS, WES, gene panel testing, and often single gene testing.
• It is a sequencing technique where hundreds of thousands of fragments of DNA are sequenced in parallel.
• Although it does not detect all types of variant, massively parallel sequencing is particularly useful in clinical situations where many possible genes and variant types could be causing a patient’s features.

For clinicians

• Association for Clinical Genetic Science: Practice guidelines for targeted next-generation sequencing analysis and interpretation (PDF, 13 pages)
• Applied Biological Materials: Next generation sequencing (NGS) – An introduction (video, 2 minutes 47 seconds)
• European Society of Human Genetics: A guide to genetic tests that are used to examine many genes at the same time (PDF, seven pages)

References:

• Muzzey D, Evans EA and Lieber C. ‘Understanding the basics of NGS: From mechanism to variant calling‘. Current Genetic Medicine Reports 2015: volume 3, issue 4, pages 158–165. DOI: 10.1007/s40142-015-0076-8
• Shendure J, Findlay GM and Snyder MW. ‘Genomic medicine – progress, pitfalls and promise‘. Cell 2019: volume 177, issue 1, pages 45–57. DOI: 10.1016/j.cell.2019.02.003