Introduction
Highly-annotated, well-sourced bio-specimens are essential in elucidating cancer initiation and progression and in validating panels for clinically actionable use. Formalin-fixed, paraffin-embedded (FFPE) tissue archives are most commonly used for oncology research and validation studies. However, accurate mutation detection is often problematic in FFPE tissue since it tends to have highly variable levels of fragmentation that limit the amount of usable DNA templates. In addition, formalin-introduced sequence artifacts and PCR inhibitors further obstruct mutation detection. The lack of consistency between the various protocols for sample handling and extraction of DNA is one of the major obstacles in translating analysis to clinical practice.
This technical note describes the development of a multiplexed panel to determine sample quality and identification (ID) using the MassARRAY® System. This panel performs DNA quality assessment and template copy number enumeration across a broad dynamic range of copies and monitors sample fragment size over a 100-500 bp range, while uniquely identifying the sample using exonic SNPs.
Assay Design
The Assays by Agena (ABA) Exome QC Panel is a set of pre-verified assays to assess DNA quality and amplifiable template copy number across a range of 100-100,000 copies (0.3-300 ng) and monitor sample fragment size between 100-500 bp. The DNA quality is assessed using 25 competitive PCR assays targeting known housekeeping genes. These genes are selected from cancer studies where they exhibit low levels of germline and somatic copy number alterations, SNPs, and somatic mutations within the coding sequence. The intact, amplifiable template copy number across a size range of 100, 200, 300, 400, and 500 nucleotides is assessed using 5 assays at each amplicon size, with 300, 1000, 3000, 10,000, or 100,000 copies of competitive template. Sample identification and matching is done via 21 exonic SNPs, with high minor allele frequency reported across major HapMart populations, along with 3 XY paralogues for gender determination. Completely automated software supports localized historical identity matching and sample quality assessment.
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