Institute on the Biology of Aging and Metabolism, University of Minnesota, Minneapolis, MN, USA.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
Nat Protoc. 2024 Feb;19(2):487-516. doi: 10.1038/s41596-023-00914-8. Epub 2023 Nov 23.
Somatic mutations are the cause of cancer and have been implicated in other, noncancerous diseases and aging. While clonally expanded mutations can be studied by deep sequencing of bulk DNA, very few somatic mutations expand clonally, and most are unique to each cell. We describe a detailed protocol for single-cell whole-genome sequencing to discover and analyze somatic mutations in tissues and organs. The protocol comprises single-cell multiple displacement amplification (SCMDA), which ensures efficiency and high fidelity in amplification, and the SCcaller software tool to call single-nucleotide variations and small insertions and deletions from the sequencing data by filtering out amplification artifacts. With SCMDA and SCcaller at its core, this protocol describes a complete procedure for the comprehensive analysis of somatic mutations in a single cell, covering (1) single-cell or nucleus isolation, (2) single-cell or nucleus whole-genome amplification, (3) library preparation and sequencing, and (4) computational analyses, including alignment, variant calling, and mutation burden estimation. Methods are also provided for mutation annotation, hotspot discovery and signature analysis. The protocol takes 12-15 h from single-cell isolation to library preparation and 3-7 d of data processing. Compared with other single-cell amplification methods or single-molecular sequencing, it provides high genomic coverage, high accuracy in single-nucleotide variation and small insertions and deletion calling from the same single-cell genome, and fewer processing steps. SCMDA and SCcaller require basic experience in molecular biology and bioinformatics. The protocol can be utilized for studying mutagenesis and genome mosaicism in normal and diseased human and animal tissues under various conditions.
体细胞突变是癌症的病因，并与其他非癌性疾病和衰老有关。虽然通过对大量 DNA 进行深度测序可以研究克隆扩增的突变，但只有极少数体细胞突变会克隆扩增，而且大多数突变都是每个细胞所特有的。我们描述了一种详细的单细胞全基因组测序方案，用于发现和分析组织和器官中的体细胞突变。该方案包括单细胞多次置换扩增（SCMDA），该方法可确保扩增的效率和高保真度，以及 SCcaller 软件工具，该工具可通过滤除扩增伪影，从测序数据中调用单核苷酸变异和小插入缺失。SCMDA 和 SCcaller 是该方案的核心，它描述了一种全面分析单细胞体细胞突变的完整程序，涵盖了（1）单细胞或细胞核分离、（2）单细胞或细胞核全基因组扩增、（3）文库制备和测序、（4）计算分析，包括比对、变异调用和突变负担估计。还提供了突变注释、热点发现和特征分析的方法。从单细胞分离到文库制备需要 12-15 小时，数据处理需要 3-7 天。与其他单细胞扩增方法或单分子测序相比，它提供了更高的基因组覆盖度、从同一单细胞基因组中更高的单核苷酸变异和小插入缺失调用准确性，以及更少的处理步骤。SCMDA 和 SCcaller 需要基本的分子生物学和生物信息学经验。该方案可用于在各种条件下研究正常和患病的人类和动物组织中的诱变和基因组镶嵌现象。
