Genome coverage and sequence fidelity of φ29 polymerase-based multiple strand displacement whole genome amplification -: art. no. e71
Genome coverage and sequence fidelity of φ29 polymerase-based multiple strand displacement whole genome amplification -: art. no. e71
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DOI：
10.1093/nar/gnh069
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发表时间：
2004-05-01
影响因子：
14.9
通讯作者：
Sellers, WR
中科院分区：
文献类型：
--
作者：
Paez, JG;Lin, M;Sellers, WR
目前正在系统地确定与疾病有因果关系的体细胞和种系遗传变异。然而，实际临床样本的全基因组遗传分析受到可用基因组DNA缺乏的限制。在这里，我们使用直接测序和高密度寡核苷酸阵列检测了基于phi29聚合酶的基因组扩增（phi29MDA）的保真度和基因组代表性，探测了bb10000个SNP等位基因。基因组的表达是全面的，估计有99.82%完成，尽管6个区域（最多5.62 Mb）未能扩增。SNP基因分型的准确性没有下降，在采样50万bp的直接测序实验中，估计错误率（9.5 x 10(-6)）与配对未扩增样本相同。检测癌症相关的杂合性缺失和拷贝数变化，包括纯合子缺失和基因扩增，同样稳健。这些结果表明，phi29MDA具有高保真度，近乎完整的基因组表达，适合高分辨率遗传分析。
Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of phi29 polymerase-based genome amplification (phi29MDA) using direct sequencing and high density oligonucleotide arrays probing >10 000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500 000 bp, the estimated error rate (9.5 x 10(-6)) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that phi29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.