Alcaide Miguel, Rushton Christopher, Morin Ryan D
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.
Methods Mol Biol. 2019;1956:383-435. doi: 10.1007/978-1-4939-9151-8_20.
Liquid biopsies are rapidly emerging as powerful tools for the early detection of cancer, noninvasive genomic profiling of localized or metastatic tumors, prompt detection of treatment resistance-associated mutations, and monitoring of therapeutic response and minimal residual disease in patients during clinical follow-up. Growing evidence strongly supports the utility of circulating tumor DNA (ctDNA) as a biomarker for the stratification and clinical management of lymphoma patients. However, ctDNA is diluted by variable amounts of cell-free DNA (cfDNA) shed by nonneoplastic cells causing a background signal of wild-type DNA that limits the sensitivity of methods that rely on DNA sequencing. Here, we describe an error suppression method for single-molecule counting that relies on targeted sequencing of cfDNA libraries constructed with semi-degenerate barcode adapters. Custom pools of biotinylated DNA baits for target enrichment can be designed to specifically track somatic mutations in one patient, survey mutation hotspots with diagnostic and prognostic value or be comprised of comprehensive gene panels with broad patient coverage in lymphoma. Such methods are amenable to track ctDNA levels during longitudinal liquid biopsy testing with high specificity and sensitivity and characterize, in real time, the genetic profiles of tumors without the need of standard invasive biopsies. The analysis of ultra-deep sequencing data according to the bioinformatics pipelines also described in this chapter affords to harness lower limits of detection for ctDNA below 0.1%.
液体活检正迅速成为癌症早期检测、局部或转移性肿瘤的非侵入性基因组分析、及时检测与治疗耐药相关的突变以及在临床随访期间监测患者治疗反应和微小残留病的有力工具。越来越多的证据有力地支持了循环肿瘤DNA（ctDNA）作为淋巴瘤患者分层和临床管理生物标志物的实用性。然而，ctDNA会被非肿瘤细胞释放的不同数量的游离DNA（cfDNA）稀释，从而产生野生型DNA的背景信号，这限制了依赖DNA测序方法的灵敏度。在此，我们描述了一种单分子计数的错误抑制方法，该方法依赖于用半简并条形码接头构建的cfDNA文库的靶向测序。用于靶标富集的生物素化DNA诱饵定制池可设计用于特异性追踪一名患者的体细胞突变、调查具有诊断和预后价值的突变热点，或由涵盖淋巴瘤广泛患者群体的综合基因panel组成。此类方法适合在纵向液体活检检测期间以高特异性和灵敏度追踪ctDNA水平，并实时表征肿瘤的基因图谱，而无需进行标准的侵入性活检。根据本章中也描述的生物信息学流程分析超深度测序数据，能够利用低于0.1%的ctDNA检测下限。
