Institute of Stem Cell Research, Helmholtz Center Munich, 85764, Neuherberg, Germany.
Genome Biology Unit, European Molecular Biology Laboratory, 69117, Heidelberg, Germany.
Genome Biol. 2019 Aug 6;20(1):155. doi: 10.1186/s13059-019-1748-6.
We describe a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts or genomic regions from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, which are all pre-selected and optimized in silico. By applying the matrices in a novel workflow named Barcode Assembly foR Targeted Sequencing (BART-Seq), we analyze developmental states of thousands of single human pluripotent stem cells, either in different maintenance media or upon Wnt/β-catenin pathway activation, which identifies the mechanisms of differentiation induction. Moreover, we apply BART-Seq to the genetic screening of breast cancer patients and identify BRCA mutations with very high precision. The processing of thousands of samples and dynamic range measurements that outperform global transcriptomics techniques makes BART-Seq first targeted sequencing technique suitable for numerous research applications.
我们描述了一种高度敏感、定量且廉价的技术，可用于同时对数千个批量样本或单细胞中的转录组或基因组区域进行靶向测序。多重化基于一种简单的方法，该方法生成大量附着在不变引物对上的不同 DNA 条码矩阵，这些条码均经过预先选择和计算机优化。通过在一种名为靶向测序条码组装（BART-Seq）的新型工作流程中应用这些矩阵，我们分析了数千个人类多能干细胞在不同维持培养基中的发育状态，或在 Wnt/β-catenin 通路激活后，确定了分化诱导的机制。此外，我们将 BART-Seq 应用于乳腺癌患者的遗传筛选，并以非常高的精度鉴定 BRCA 突变。该技术可处理数千个样本并进行动态范围测量，其性能优于全球转录组学技术，使 BART-Seq 成为首个适合众多研究应用的靶向测序技术。
