Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples
Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples
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DOI：
10.1101/gr.101212.109
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发表时间：
2010-09-01
期刊：
影响因子：
7
通讯作者：
Mitra, Robi David
中科院分区：
文献类型：
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作者：
Varley, Katherine Elena;Mitra, Robi David
在癌症进展过程中，异常DNA甲基化经常发生在基因启动子处。识别这些基因座很重要，因为它们经常被错误调节并驱动肿瘤发生。亚硫酸氢盐测序是用于鉴定异常启动子甲基化的最直接和最高分辨率的测定。最近，基因组捕获方法已与下一代测序相结合，以实现个体样品中甲基化的基因组规模调查。然而，验证通过这些方法鉴定的候选基因座是具有挑战性的，因为不存在对大量样品中超过50个差异甲基化基因座进行亚硫酸氢盐测序的有效方法。为了解决这个问题，我们开发了亚硫酸氢盐补丁PCR，它可以在许多样品中进行高度多重的亚硫酸氢盐PCR和测序。利用该方法，我们成功地在同一反应中同时扩增了94个靶基因启动子的100%。通过将样品特异性DNA条形码并入扩增子中，我们在454 Life Sciences（Roche）FLX测序仪的单次运行中分析了48个样品。该方法需要少量的起始DNA（250 ng），并且不需要鸟枪文库构建。该方法具有高度特异性; 90%的测序读数与靶基因座对齐。靶向启动子来自乳腺癌和结肠癌中经常突变的基因，样本包括乳腺癌和结肠癌以及邻近的正常组织。这种方法使我们能够鉴定出9种基因启动子，它们表现出在结肠癌和乳腺癌中经常发生的肿瘤特异性DNA甲基化缺陷。我们还分析了单核苷酸多态性，以观察肿瘤发生过程中特定等位基因上积累的DNA甲基化。该方法广泛适用于使用下一代测序研究大量患者样本中的DNA甲基化。
Aberrant DNA methylation frequently occurs at gene promoters during cancer progression. It is important to identify these loci because they are often misregulated and drive tumorigenesis. Bisulfite sequencing is the most direct and highest resolution assay for identifying aberrant promoter methylation. Recently, genomic capture methods have been combined with next-generation sequencing to enable genome-scale surveys of methylation in individual samples. However, it is challenging to validate candidate loci identified by these approaches because an efficient method to bisulfite sequence more than 50 differentially methylated loci across a large number of samples does not exist. To address this problem, we developed Bisulfite Patch PCR, which enables highly multiplexed bisulfite PCR and sequencing across many samples. Using this method, we successfully amplified 100% of 94 targeted gene promoters simultaneously in the same reaction. By incorporating sample-specific DNA barcodes into the amplicons, we analyzed 48 samples in a single run of the 454 Life Sciences (Roche) FLX sequencer. The method requires small amounts of starting DNA (250 ng) and does not require a shotgun library construction. The method was highly specific; 90% of sequencing reads aligned to targeted loci. The targeted promoters were from genes that are frequently mutated in breast and colon cancer, and the samples included breast and colon tumor and adjacent normal tissue. This approach allowed us to identify nine gene promoters that exhibit tumor-specific DNA methylation defects that occur frequently in colon and breast cancer. We also analyzed single nucleotide polymorphisms to observe DNA methylation that accumulated on specific alleles during tumor development. This method is broadly applicable for studying DNA methylation across large numbers of patient samples using next-generation sequencing.