Introduction
Targeted Sequencing (TS) Methodology: Precision Profiling of Clinically-Relevant Genomic Regions
The TS methodology meticulously investigates specific genomic regions, frequently encompassing numerous well-established drivers or clinically actionable genes of interest. This approach is designed to identify sequence variants with a high level of confidence and accuracy. Illustratively, genes such as KRAS and TP53, renowned for their recurrent mutations at various hotspots, are commonly targeted across diverse cancer types. Similarly, BRAF and EGFR are systematically screened in a spectrum of solid tumors due to their harboring of clinically significant mutations. This strategic targeting, as elucidated by Bewicke-Copley (2019), underscores the precision and relevance of the TS approach in elucidating genetic alterations associated with various cancers.
Applications
A substantial body of clinical investigations employs targeted panels in the range of 25 to 122 genes, utilizing genomic Targeted Sequencing (TS) for the analysis of clinical cancer samples. Notable cancers subjected to this approach encompass Acute Myeloid Leukemia, Breast Cancer, Pancreatic Cancer, and Skin Cancer, as expounded by Bewicke-Copley (2019). Beyond the oncological domain, Targeted Region Sequencing finds applicability in various areas, including:
– Human population studies
– Linkage analysis for inherited diseases
– Detection and quantification of low-frequency alleles and rare variants
– Identification of somatic and germline mutations
– Exploration for biomarkers and therapeutic targets
Benefits
Targeted Region Sequencing (TS) stands as a robust and versatile tool for the comprehensive profiling of clinical samples in the domains of cancer research and clinical trials. The substantial sequencing depth employed in TS, exemplified by ultra-deep sequencing at a magnitude of 10,000x and beyond, renders it particularly potent for the profiling of clinical samples, including those derived from formalin-fixed paraffin-embedded (FFPE) tissues and circulating tumor DNA (ctDNA), where DNA quality and/or tumor content may be limited.
The heightened depth of coverage characteristic of TS facilitates the identification of mutations existing in minute fractions of malignant cells, known as sub-clonal mutations. In the context of detecting minimal residual disease, TS achieves variant allele frequency (VAF) detection as low as 0.1–0.2%. These attributes collectively position TS as superior to non-Next Generation Sequencing (NGS) based techniques, such as Sanger sequencing and digital polymerase chain reaction (PCR), as well as Whole Genome Sequencing (WGS) and Whole Exome Sequencing (WES), particularly in the context of large-scale genomic testing and clinical trial applications (Bewicke-Copley, 2019).
Commercially accessible targeted gene panels, typically tailored for research or clinical applications, are specifically crafted to amplify genomic regions that are recognized as pertinent within the context of cancer or specific cancer subtypes. The utilization of these panels expedites the sequencing process substantially, given that they have undergone prior design, rigorous testing, and validation, as elucidated by Bewicke-Copley (2019).
Sample Requirements
Sample Specifications
In this context, the term ‘targeted panel’ denotes a set of genomic coordinates curated based on user interest. A significant distinction between Whole Exome Sequencing (WES) panels and targeted panels lies in the fact that Targeted Sequencing (TS) is not restricted to canonical gene targets; it has the capability to focus on alternative regions, including promoters or breakpoints, as outlined by Bewicke-Copley (2019).