High-throughput development and characterization of a genomewide collection of gene-based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

We describe here a system for the rapid identification, assay development, and characterization of gene-based single nucleotide polymorphisms (SNPs). This system couples informatics tools that mine candidate SNPs from public expressed sequence tag resources and automatically designs assay reagents with detection by a chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry platform. As a proof of concept of this system, a genomewide collection of reagents for 9,115 gene-based SNP genetic markers was rapidly developed and validated. These data provide preliminary insights into patterns of polymorphism in a genomewide collection of gene-based polymorphisms.

Figures

Figure 1

Scheme of the analytical process to confirm and validate SNPs using chip-based mass spectrometry. After setup of an equimolar mixture of 94 individual Centre d'Étude du Polymorphisme Humain DNA samples, gene-specific uniplex PCRs were performed. The amplicon pool was subjected to a post-PCR primer extension reaction (massextend). Nanoliter amounts of the extension products were loaded onto SpectroCHIPs and subsequently analyzed by an array mass spectrometer.

Figure 2

Example spectra representing the range of assay qualities. Allelic peaks are indicated at the top of each spectrum. (A) Low-end assay quality, total peak area of both alleles (pa; arbitrary units) = 768, signal-to-noise ratio (snr) = 24.7, relative frequency for C allele = 0.23, T allele = 0.77, standard deviation (SD) of four individual spottings onto SpectroCHIPs followed by mass spectrometric analysis = 0.034. (B) pa = 1,995, snr = 33.3, C allele = 0.37, T allele = 0.63, SD = 0.012. (C) Average assay quality, pa = 9,957, snr = 77.4, C allele = 0.55, T allele = 0.45, SD = 0.009. (D) High-end assay quality, pa = 48,679, snr = 199, C allele = 0.77, T allele = 0.23, SD = 0.006.

Figure 3

Allelic frequency range of 3,646 SNP genetic markers with a frequency in the Centre d'Étude du Polymorphisme Humain population greater than 10%.