Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer.
Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer.
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DOI：
10.1093/dnares/dss021
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发表时间：
2012-10
期刊：
影响因子：
--
通讯作者：
Vasmatzis G
中科院分区：
文献类型：
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作者：
Murphy SJ;Cheville JC;Zarei S;Johnson SH;Sikkink RA;Kosari F;Feldman AL;Eckloff BW;Karnes RJ;Vasmatzis G
高通量下一代测序提供了一个革命性的平台，可以解开隐藏在肿瘤细胞亚群中的精确DNA畸变。然而，在许多情况下，有限的细胞数量使得该技术在肿瘤异质性研究中的应用成为一个挑战。为了解决这些局限性，我们提出了一种新的方法，通过直接在LCM移植细胞上原位进行的全基因组扩增（WGA）协议，将激光捕获显微切割（LCM）与测序平台结合起来。我们进一步调整了目前的Illumina配对（MP）测序方案，以输入WGA DNA，并使用该技术研究了由LCM分别收集的相邻Gleason模式3和4前列腺肿瘤中的大基因组重排。测序数据预测的基因组覆盖度和深度类似于未扩增的基因组DNA，在WGA方案中预测的重复和偏差有限。我们实验室开发的映射算法预测了高置信度的重排，并且选定的事件在验证后各自证明了预测的融合连接。在未扩增的组织中额外确认重排，并在相邻的良性组织中进行评价。对表征癌症的基因融合的详细理解将在生物标志物的开发中至关重要，以预测临床结果。所描述的方法提供了一种在有限的纯肿瘤组织群体中有效定义这些事件的机制，有助于衍生引发癌症和驱动癌症进展的基因组畸变。
High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.
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影响因子：
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作者：
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