NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR.
NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR.
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DOI：
10.1093/nar/gkaa1175
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发表时间：
2021-02-26
影响因子：
14.9
通讯作者：
Makrigiorgos GM
中科院分区：
文献类型：
--
作者：
Yu F;Leong KW;Makrigiorgos A;Adalsteinsson VA;Ladas I;Ng K;Mamon H;Makrigiorgos GM
使用下一代测序技术(NGS)对组织或液体活检中的微卫星不稳定性(MSI)进行灵敏的检测在癌症预后和预测方面的应用日益广泛。然而，NGS的复杂性使其与已建立的MSI多重PCR检测相比变得繁琐。我们提出了一种新的检测MSI的方法，该方法结合了多重PCR的实际优势和NGS提供的信息广度。Inter-Alu-PCR利用存在于每个Alu元件中的可变长度的多聚腺嘌呤重复序列，提供了一种大规模并行的快速方法，可以在从相邻Alu序列产生的短80-150bp的扩增片段中捕获富含PolyA的基因组片段。定制的软件分析工具MSI-Tracer能够从组织活检中检测Alu相关的MSI，或在循环DNA中进行低水平的MSI追踪。根据匹配的正常组织的可用性和不稳定的程度，可以检测到体细胞频率为0.05-1.5%的MSI相关INDELs。由于人类基因组中的Alu拷贝数很高，一次间歇Alu-PCR法可以从∼100pg循环DNA中检索足够的信息来识别MSI相关的Indels，将电流限制减少了∼2个数量级，相当于从手指上获得的循环DNA。Alu间聚合酶链式反应的实用和信息优势使其成为在液体活检中识别组织MSI状态或追踪MSI相关指标的有力工具。
Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new approach to detect MSI using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed-PCR with the breadth of information provided by NGS. Inter-Alu-PCR employs poly-adenine repeats of variable length present in every Alu element and provides a massively-parallel, rapid approach to capture poly-A-rich genomic fractions within short 80–150bp amplicons generated from adjacent Alu-sequences. A custom-made software analysis tool, MSI-tracer, enables Alu-associated MSI detection from tissue biopsies or MSI-tracing at low-levels in circulating-DNA. MSI-associated indels at somatic-indel frequencies of 0.05–1.5% can be detected depending on the availability of matching normal tissue and the extent of instability. Due to the high Alu copy-number in human genomes, a single inter-Alu-PCR retrieves enough information for identification of MSI-associated-indels from ∼100 pg circulating-DNA, reducing current limits by ∼2-orders of magnitude and equivalent to circulating-DNA obtained from finger-sticks. The combined practical and informational advantages of inter-Alu-PCR make it a powerful tool for identifying tissue-MSI-status or tracing MSI-associated-indels in liquid biopsies.
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