The Genom of Homo sapiens.pdf

76 FAN ET AL.Figure 7. Allele-specific expression monitoring. Both genomic DNA and total RNA (converted to cDNA) from an ovarian tissue samplewere genotyped for 32 SNPs in gene coding regions. Arrows indicate SNPs, called as heterozygous in gDNA, which show a clearallelic imbalance in mRNA abundance.showed a strong allele-specific bias in transcript abundance.In addition, assays for 1,152 cSNPs from 450 humancancer-related genes were monitored in pairs ofDNA/RNA samples isolated from different human tissues.Of the cSNPs (genes) that were expressed at detectablelevels in the RNA and heterozygous in the correspondinggenomic DNA, 20% showed differences inallelic abundance at 95% confidence; in many cases, onlyone allele from a heterozygous genomic locus was detected(J.-B. Fan, unpubl.). These preliminary studiesshow the feasibility of using the GoldenGate assay togenotype cDNA derived from mRNA populations.CONCLUSIONSWe have developed a flexible, accurate, and scalablegenotyping system, and have achieved high accuracy togetherwith high call rates. Conventional wisdom wasthat single-plex assays would be more accurate thanhighly multiplexed assays, and that it would be difficultto optimize assays in a multiplex format. In fact, our accuracyand call rates are similar to those obtained fromsingle-plex genotyping systems, but at over 1,000 timesthe assay sequence complexity. These results demonstratethe specificity of the GoldenGate assay format, aswell as the reproducibility and accuracy of the BeadArrayplatform and the BeadLab genotyping system as a whole.We previously published a ligation-based RNA assayfor analysis of mRNA splice variants (Yeakley et al.2002). In the course of this work, others have also publishedgenotyping assays that are conceptually similar(Schouten et al. 2002; Hardenbol et al. 2003), and othershave also achieved high multiplexing levels using directhybridization-based methods, albeit without the flexibilityto assay any SNPs of interest (Kennedy et al. 2003).The assay format used by Schouten et al. (2002), which isread out using gel electrophoresis, is used for the analysisof DNA copy number variation, including the detectionof deletions in gDNA.The GoldenGate assay is versatile and can be adaptedto a variety of other applications, such as methylationprofiling (J.-B. Fan, unpubl.). In addition, since we use auniversal address scheme, different address sequencescan be assigned to the same SNP locus to interrogate thesame SNP in different samples. This strategy can be quiteuseful for studies that involve many samples but relativelyfew SNPs, as in some plant and animal breedingapplications. We tested this scheme with an experimentaldesign in which a set of 96 SNPs was associated with 10discrete sets of 96 address sequences and used to genotype10 samples in parallel, with readout on one array.We obtained exactly the same genotyping results usingthis pooling scheme as with our standard approach.In conclusion, we believe that the GoldenGate assayformat is an exemplar for a new class of highly multiplexedassays that utilize parallel readout systems. It representsa significant departure from single and low-multiplexassays and is well suited for large-scale analysis ofcomplex biological systems. We expect that large-scalegenotyping studies using this approach will help eluci-