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题目:: Single Cell Whole Genome Amplification Technique Impacts the Accuracy of SNP Microarray Based Genotyping and Copy Number Analyses
ID:
状态:: 发布时间Dec. 20, 2010 , 更新时间 May 2, 2014
物种:: Homo sapiens
摘要:: Methods of comprehensive microarray based analyses of single cell DNA are rapidly emerging. Whole genome amplification (WGA) remains a critical component for these methods to be successful. A number of commercially available WGA kits have been independently utilized in previous single cell microarray studies. However, direct comparison of their performance on single cells has not been conducted. The present study demonstrates that among previously published methods, a single cell GenomePlex WGA protocol provides the best combination of speed and accuracy for SNP microarray based copy number analysis when compared to a REPLI-g or GenomiPhi based protocol. Alternatively, for applications that do not have constraints on turn-around time and that are directed at accurate genotyping rather than copy number assignments, a REPLI-g based protocol may provide the best solution. Affymetrix SNP arrays were processed according to the manufacturer's directions on DNA extracted from human fibroblast cell lines and single fibroblast cells. Afflymetrix SNP array analysis was successfully completed on 46 lymphocyte single cell samples, 8 gDNA extracted from cell lines, 11 reference gDNA extracted from cell lines and 3 reference gDNA samples from the RMA of New Jersey DNA bank. GSM617116 to GSM617129: CEL files were processed using GTYPE version 4 (Affymetrix Inc., Genotyping Console 4.0 Manual) using the DM algorithm for genotype calls. Copy number and loss of heterozygosity were calculated from CHP files using CNAT version 4.1 (Affymetrix Inc., Genotyping Console 4.0 Manual) analysis against a reference set consisting of three normal females from in house gDNA bank, 11 normal females from Coriel cell lines and 16 normal females from the HapMap database (www.hapmap.org). The 16 normal females are NA10855, NA10863, NA11832, NA12057, NA12234, NA12717, NA12813, NA18505, NA18508, NA18517, NA19137, NA19152, NE00088, NE00091, NE00403, and NE01119.
实验种类:: comparative genomic hybridization by array
样本量:: 56
实验设计:
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数据号:: E-GEOD-24690, GSE24690
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