Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers

Targeted Next Generation Sequencing (NGS) is being adopted increasingly broadly in many research, commercial and clinical settings. Currently used target capture methods, however, typically require complex and lengthy (sometimes multi-day) workflows that complicates their use in certain applications. In addition, small panels for high sequencing depth applications such as liquid biopsy typically have low on-target rates, resulting in unnecessarily high sequencing cost. We have developed a novel targeted sequencing library preparation method, named Linked Target Capture (LTC), which replaces typical multi-day target capture workflows with a single-day, combined ‘target-capture-PCR’ workflow. This approach uses physically linked capture probes and PCR primers and is expected to work with panel sizes from 100 bp to >10 Mbp. It reduces the time and complexity of the capture workflow, eliminates long hybridization and wash steps and enables rapid library construction and target capture. High on-target read fractions are achievable due to repeated sequence selection in the target-capture-PCR step, thus lowering sequencing cost. We have demonstrated this technology on sample types including cell-free DNA (cfDNA) and formalin-fixed, paraffin-embedded (FFPE) derived DNA, capturing a 35-gene pan-cancer panel, and therein detecting single nucleotide variants, copy number variants, insertions, deletions and gene fusions. With the integration of unique molecular identifiers (UMIs), variants as low as 0.25% abundance were detected, limited by input mass and sequencing depth. Additionally, sequencing libraries were prepared in less than eight hours from extracted DNA to loaded sequencer, demonstrating that LTC holds promise as a broadly applicable tool for rapid, cost-effective and high performance targeted sequencing.靶向下一代测序（Next Generation Sequencing，NGS）正日益广泛地应用于诸多科研、商业及临床场景中。然而，当前常用的靶向捕获（target capture）方法通常需要复杂且耗时（有时长达数天）的实验流程，这增加了其在部分应用场景中的使用难度。此外，针对液体活检等高测序深度应用场景的小型靶向测序panel，通常靶标上率较低，导致测序成本不必要地升高。本研究开发了一种新型靶向测序文库制备方法，命名为连接式靶向捕获（Linked Target Capture，LTC），该方法将传统耗时数天的靶向捕获流程整合为单日完成的‘靶向捕获-聚合酶链式反应（Polymerase Chain Reaction，PCR）’联合流程。该方法采用物理连接的捕获探针与PCR引物，适配的靶向测序panel大小范围为100碱基对（bp）至10兆碱基对（Mbp）以上。该方法缩短了靶向捕获流程的耗时与复杂度，省去了冗长的杂交与洗涤步骤，可实现快速的文库构建与靶向捕获。由于在靶向捕获-PCR步骤中可实现多轮序列筛选，因此可获得较高的靶标上测序读段比例，进而降低测序成本。本研究已在多种样本类型中验证了该技术的有效性，包括循环游离DNA（cell-free DNA，cfDNA）与福尔马林固定石蜡包埋（formalin-fixed, paraffin-embedded，FFPE）来源的DNA；我们利用该方法捕获了包含35个基因的泛癌检测panel，并成功检出单核苷酸变异、拷贝数变异、插入变异、缺失变异及基因融合事件。通过整合唯一分子标识符（unique molecular identifiers，UMIs），本方法可检出丰度低至0.25%的变异，该检测下限受起始样本投入量与测序深度的限制。此外，从提取DNA到将测序文库加载至测序仪的整个流程可在8小时内完成，这表明连接式靶向捕获（LTC）有望成为一款应用广泛、快速高效且成本低廉的高性能靶向测序工具。