High-throughput, high-accuracy array-based resequencing

Affymetrix Inc., 3420 Central Expressway, Santa Clara, CA 95051, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2009; 106(16):6712-7. DOI: 10.1073/pnas.0901902106
Source: PubMed


Although genomewide association studies have successfully identified associations of many common single-nucleotide polymorphisms (SNPs) with common diseases, the SNPs implicated so far account for only a small proportion of the genetic variability of tested diseases. It has been suggested that common diseases may often be caused by rare alleles missed by genomewide association studies. To identify these rare alleles we need high-throughput, high-accuracy resequencing technologies. Although array-based genotyping has allowed genomewide association studies of common SNPs in tens of thousands of samples, array-based resequencing has been limited for 2 main reasons: the lack of a fully multiplexed pipeline for high-throughput sample processing, and failure to achieve sufficient performance. We have recently solved both of these problems and created a fully multiplexed high-throughput pipeline that results in high-quality data. The pipeline consists of target amplification from genomic DNA, followed by allele enrichment to generate pools of purified variant (or nonvariant) DNA and ends with interrogation of purified DNA on resequencing arrays. We have used this pipeline to resequence approximately 5 Mb of DNA (on 3 arrays) corresponding to the exons of 1,500 genes in >473 samples; in total >2,350 Mb were sequenced. In the context of this large-scale study we obtained a false positive rate of approximately 1 in 500,000 bp and a false negative rate of approximately 10%.

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    • "Affordability is at present the main barrier and as a medium-term solution enrichment of individual chromosomal regions containing GLRA1 and SLC6A5 may provide an interim solution. For example, Zheng and colleagues have recently developed a method for high-throughput variant detection, utilising specific genomic regions for target amplification by capture and ligation (TACL), allele enrichment and array resequencing (Zheng et al., 2009). This platform has identified rare and novel variants, and will undoubtedly lead to improvements in our understanding of complex genetic disorders. "
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    • "Due to the use of tiling probes in resequencing arrays, a single nucleotide mutation at a particular query base could cause a dramatic reduction in the hybridization intensities of neighbouring PM probes up to six bases away (14). This effect can be measured by studying the NHIP of each query base. "
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