A new wave of automated, inexpensive, and high-throughput multiplexing technologies has been widely reported on of late. Diagnostic laboratories and clinical pharmacologists will find these multiplexing systems ideal for identifying and validating biomarkers for diverse applications ranging from testing for adverse drug reactions (ADR) to prognostics and diagnostics. In this tutorial, we discuss the application of one of these multiplex technologies for validating DNA biomarkers.
The use of highly multiplexed genotyping arrays to interrogate several hundred thousand single nucleotide polymorphisms (SNPs) per sample has made possible significant leaps in the understanding of the human genome.
Such genome-scale solutions are particularly effective for initial discovery work where the goal is to begin the process of correlating gene mutations with disease phenotypes. This is achieved by comparing SNPs across hundreds to thousands of samples obtained from both healthy and diseased sources.
Since there are more SNPs in the human genome than is currently possible to assay on one chip, Illumina (www.illumina.com) uses tag SNPs representing specific regions of the genome rather than one individual mutation. The initial discovery work using highly multiplexed arrays may identify up to several hundred tag SNPs.
To identify the precise biomarkers associated with disease, researchers must map DNA regions and characterize many more SNPs per region. At this stage, there are far fewer targets analyzed per sample, but more samples are analyzed overall. Because of the focused analysis on fewer targets per sample, highly multiplexed systems are no longer cost-effective, and the cost emphasis changes from striving for the lowest cost per SNP to seeking the lowest cost per sample.
Conversely, single-plex technologies may be quick and inexpensive for individual targets but become too costly when applied to many targets per sample. Thus there is a growing need for technologies like Illumina’s VeraCode™, which can analyze one to several hundred targets per sample.
Biomarker validation is the confirmatory step in determining whether a particular mutation is associated with a disease. At this point, thousands of samples from both healthy and diseased sources are screened for the relevant biomarkers. In addition to the constraints mentioned previously, the data generated during biomarker validation must be reliable and reproducible if SNPs are to be used later to aid in the diagnosis of a particular disease or for guiding prognosis of disease outcome.
Illumina’s VeraCode technology specifically addresses the need for a low- to mid-level multiplexing, high throughput, and low cost-per-sample instrument capable of processing the focused assays necessary for biomarker validation.
At the heart of VeraCode technology are cylindrical glass beads measuring 240 microns in length by 28 microns in diameter. Each VeraCode microbead is inscribed with a unique digital holographic code to designate and track the specific analyte or genotype of interest (Figure).