Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.
Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.
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DOI：
10.1186/1471-2164-7-216
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发表时间：
2006-08-23
期刊：
影响因子：
4.4
通讯作者：
Leamon JH
中科院分区：
文献类型：
--
作者：
Pinard R;de Winter A;Sarkis GJ;Gerstein MB;Tartaro KR;Plant RN;Egholm M;Rothberg JM;Leamon JH
全基因组扩增是一种越来越普遍的技术，通过这种技术，微量的DNA可以倍增，以产生适合基因检测和分析的数量。由这些方法产生的扩增引起的误差和模板偏差的问题先前已经通过小规模（SNP）或大规模（CGH阵列，FISH）方法来解决。在这里，我们利用全基因组测序来评估两种细菌基因组的编码区和非编码区中扩增诱导的偏倚。通过几种常见的市售方案扩增盐杆菌属NRC-1 DNA和空肠弯曲杆菌：多重置换扩增、引物延伸预扩增和简并寡核苷酸引物PCR。通过使用454测序系统技术对两种基因组进行整体测序，并将结果与未扩增对照的结果进行比较，评估每种方法的扩增诱导偏倚。所有扩增方法均诱导相对于未扩增对照的统计学显著偏倚。对于在100个碱基分辨率下评估的盐杆菌属物种NRC-1基因组，来自GenomiPhi扩增的材料的D-统计量比来自未扩增的材料的D-统计量大119倍，对于d-G大164.0倍，对于PEP-PCR大165.0倍，并且对于DOP-PCR比未扩增的对照大252.0倍。对于空肠弯曲杆菌，也在100碱基分辨率下分析，来自GenomiPhi扩增的材料的D-统计量比来自未扩增的材料的D-统计量大15倍，对于d-G大19.8倍，对于PEP-PCR大61.8倍，并且对于DOP-PCR比未扩增的对照大220.5倍。在本文研究的扩增方法中，多重置换扩增产物产生的偏差最小，并产生显着更高的扩增DNA的产量。
Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs) or large scale (CGH array, FISH) methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls. All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR. Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.
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