Reliability of high-throughput genotyping of whole genome amplified DNA in SNP genotyping studies
Reliability of high-throughput genotyping of whole genome amplified DNA in SNP genotyping studies
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DOI：
10.1002/elps.200600674
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发表时间：
2007-08-01
期刊：
影响因子：
2.9
通讯作者：
Smith, Michael W.
中科院分区：
文献类型：
--
作者：
Berthier-Schaad, Yvette;Kao, Wen Hong Linda;Smith, Michael W.
DNA的全基因组扩增（wga）正在许多实验室广泛实施，以延长只有有限数量的遗传分析样本的寿命。我们在来自同一个体的三组重复中确定了wgaDNA基因型的可靠性：(i) 23对基因组DNA (gDNA), (ii) 43对gDNA与wgaDNA对照，以及（iii） 29对独立扩增的wgaDNA。采用多位移扩增（MDA）进行扩增。在单个Illumina面板上，对164个心血管候选基因的1534个snp中的1268个进行了两种DNA类型的基因分型分析。用基因组材料成功分型的77个snp（6%）扩增DNA失败。测定SNP呼叫成功率、配对间的一致性和kappa统计量(K)。gdna - wgdna对共有54 110个基因型可用于一致性分析。gdna - wgdna对的平均K值为0.99。gDNA-wgaDNA对之间的一致性高于wgaDNA对之间的一致性（29对独立扩增的wgaDNA对的平均K为0.95，四分位数范围为0.93-1.00）。对那些无法从扩增DNA中分型的单核苷酸多态性进行统计分析，只表明这些位点更可能靠近端粒，并且在局部富含gc的序列中。总之，MDA方法产生的wgdna样品可以使用高通量技术进行基因分型，对原始DNA具有非常高的重现性，但调用率略低。DNA扩增方法为当前和未来的大规模遗传分析提供了有用的解决方案，特别是有限数量的样品和DNA。
Whole genome amplification (wga) of DNA is being widely implemented in many laboratories to extend the life of samples only available in limited quantities for genetic analysis. We determined the reliability of wgaDNA genotypes in three sets of replicates from the same individuals: (i) 23 pairs of genomic DNA (gDNA), (ii) 43 pairs gDNA versus wgaDNA, and (iii) 29 pairs of independently amplified wgaDNA. Amplification was performed using multiple displacement amplification (MDA). Genotyping was successful for both DNA types for 1268 out of 1534 SNPs from 164 cardiovascular candidate genes assayed in a single Illumina panel. Amplified DNA failed for 77 SNPs (6%) that were genotyped successfully with genomic material. Percent of successful SNP calls, and concordance between pairs and kappa statistics (K) were determined. A total of 54 110 genotypes from gDNA-wgaDNA pairs were available for concordance analysis. Mean K for gDNA-wgaDNA pairs was 0.99. Concordance between gDNA-wgaDNA pairs was higher than amongst wgaDNA pairs (mean K for the 29 independently amplified pairs of wgaDNA was 0.95; interquartile range: 0.93-1.00). A statistical analysis of those SNPs which failed to genotype from amplified DNA only revealed that those loci were more likely to be closer to the telomeres and in locally GC-rich sequences. In summary, the MDA method produces wgaDNA samples that can be genotyped using high-throughput technology with a very high reproducibility to the original DNA but with slightly lower call rates. DNA amplification methodologies provide a useful solution for current and future large-scale genetic analyses especially with limited quantities of samples and DNA.