Detection of chromosomal structural alterations in single cells by SNP arrays: a systematic survey of amplification bias and optimized workflow.
Detection of chromosomal structural alterations in single cells by SNP arrays: a systematic survey of amplification bias and optimized workflow.
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通过SNP阵列检测单细胞中染色体结构的改变：放大偏置和优化工作流程的系统调查。
DOI：
10.1371/journal.pone.0001306
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发表时间：
2007-12-12
期刊：
影响因子：
3.7
通讯作者：
Kato T
中科院分区：
文献类型：
--
作者：
Iwamoto K;Bundo M;Ueda J;Nakano Y;Ukai W;Hashimoto E;Saito T;Kato T
在使用全基因组扩增产物进行单细胞人类基因组分析时，涉及等位基因丢失和优先扩增的强烈的扩增偏差阻碍了结果的质量。利用寡核苷酸单核苷酸多态(SNP)阵列，我们系统地研究了这种扩增偏差的性质，包括频率、程度和对基因组位置的偏好，并评估了这种扩增偏差对后续的基因型和染色体拷贝数分析的影响。我们发现多重置换扩增(MDA)扩增产物的扩增偏差有很大的变异性，这种偏差严重影响了基因分型和染色体拷贝数的分析。我们为高质量扩增产物的预筛选、处理阵列数据和分析染色体结构变化建立了最佳的实验条件。使用这个优化的方案，我们成功地从一个淋巴母细胞系的单个细胞中检测到了以前未知的染色体结构变化。这些改变随后被核型分析证实。此外，我们成功地从具有复杂核型的细胞系中获得了可重复的单个细胞的染色体拷贝数图谱，表明了我们优化的工作流程的适用性和潜力。我们的结果表明，在将扩增产物用于基因组分析之前，应该对其质量进行严格的评估。这里描述的基于丙二醛的全基因组扩增和SNP阵列分析的方法将有助于探索单细胞的染色体变化。
In single-cell human genome analysis using whole-genome amplified product, a strong amplification bias involving allele dropout and preferential amplification hampers the quality of results. Using an oligonucleotide single nucleotide polymorphism (SNP) array, we systematically examined the nature of this amplification bias, including frequency, degree, and preference for genomic location, and we assessed the effects of this amplification bias on subsequent genotype and chromosomal copy number analyses. We found a large variability in amplification bias among the amplified products obtained by multiple displacement amplification (MDA), and this bias had a severe effect on the genotype and chromosomal copy number analyses. We established optimal experimental conditions for pre-screening for high-quality amplified products, processing array data, and analyzing chromosomal structural alterations. Using this optimized protocol, we successfully detected previously unidentified chromosomal structural alterations in single cells from a lymphoblastoid cell line. These alterations were subsequently confirmed by karyotype analysis. In addition, we successfully obtained reproducible chromosomal copy number profiles of single cells from the cell line with a complex karyotype, indicating the applicability and potential of our optimized workflow. Our results suggest that the quality of amplification products should be critically assessed before using them for genomic analyses. The method of MDA-based whole-genome amplification followed by SNP array analysis described here will be useful for exploring chromosomal alterations in single cells.
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影响因子：
4.5
作者：
Marcy, Yann;Ishoey, Thomas;Lasken, Roger S.;Stockwell, Timothy B.;Walenz, Brian P.;Halpern, Aaron L.;Beeson, Karen Y.;Goldberg, Susanne M. D.;Quake, Stephen R.
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Quake, Stephen R.
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通讯作者：
Gage, Fred H.
影响因子：
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通讯作者：
Tolle, R