Single gene sequencing

When is single gene sequencing done?

Single gene sequencing may be undertaken when a patient’s features are strongly indicative of a genetic condition caused by variants in a single gene (for example, the RB1 gene in hereditary retinoblastoma). In this context, it may provide a quick and cost effective molecular diagnosis.

How is single gene sequencing done?

Single gene sequencing is commonly undertaken by Sanger sequencing. As costs have come down, however, next generation sequencing (NGS) of single genes is increasingly offered by clinical diagnostic laboratories, especially for large genes.

Advantages

• Can provide rapid genetic confirmation of a diagnosis if the patient’s features are sufficiently specific.
• Can be cost-effective.
• Focusing on a single gene means that there are fewer variants requiring clinical interpretation, and less chance of identifying variants of unknown significance (VUS).
• There is no possibility of incidental findings.
• Where NGS is used, exon-level deletions and duplications may be detected alongside single nucleotide variants.
• Sanger sequencing of a single gene may be the only option for genes that are difficult to sequence by NGS, for example those with pseudogenes or those with a high ‘GC’ nucleotide content.

Limitations

• If the initial clinical suspicion is incorrect, sequential requesting of multiple single gene tests can lead to a delay to patient diagnosis (prolonging the patient’s ‘diagnostic odyssey’) compared to screening a broader panel of genes in one go.
• Sequential requesting of multiple single gene tests is not cost effective.
• Where Sanger sequencing is used, a separate assay (for example, MLPA) is required to detect exon-level deletions and duplications, where appropriate.
• Not suitable for genetically heterogeneous conditions.
• Will not identify any novel genetic causes of disease.

For a summary table comparing the advantages and disadvantages of the different approaches to gene sequencing (gene panel/whole exome sequencing/whole genome sequencing), view the Knowledge Hub article, Different approaches to gene sequencing.

• Single gene sequencing may be undertaken when a patient’s features are strongly indicative of a genetic condition caused by variants in a single gene.
• If the patient’s features are sufficiently specific, single gene sequencing can provide rapid cost-effective genetic confirmation of a diagnosis.
• Single gene sequencing is most commonly undertaken by Sanger sequencing, but it might also be carried out using massively parallel sequencing.

For clinicians

• Association for Clinical Genetic Science: Practice guidelines for Sanger Sequencing Analysis and Interpretation (PDF, six pages)

References:

• Muzzey D, Evans EA, Lieber C. ‘Understanding the basics of NGS: From Mechanism to Variant Calling‘. Current Genetic Medicine Report 2015: volume 3, issue 4, pages 158–165. DOI: 10.1007/s40142-015-0076-8