Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), National Research Council, Via Giovanni Amendola 118, 70126 Bari, Italy.
Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari "A. Moro", Via Orabona 4, 70125 Bari, Italy.
Genes (Basel). 2020 May 11;11(5):534. doi: 10.3390/genes11050534.
Mitochondria host multiple copies of their own small circular genome that has been extensively studied to trace the evolution of the modern eukaryotic cell and discover important mutations linked to inherited diseases. Whole genome and exome sequencing have enabled the study of mtDNA in a large number of samples and experimental conditions at single nucleotide resolution, allowing the deciphering of the relationship between inherited mutations and phenotypes and the identification of acquired mtDNA mutations in classical mitochondrial diseases as well as in chronic disorders, ageing and cancer. By applying an computational pipeline based on our MToolBox software, we reconstructed mtDNA genomes in single cells using whole genome and exome sequencing data obtained by different amplification methodologies (eWGA, DOP-PCR, MALBAC, MDA) as well as data from single cell Assay for Transposase Accessible Chromatin with high-throughput sequencing (scATAC-seq) in which mtDNA sequences are expected as a byproduct of the technology. We show that assembled mtDNAs, with the exception of those reconstructed by MALBAC and DOP-PCR methods, are quite uniform and suitable for genomic investigations, enabling the study of various biological processes related to cellular heterogeneity such as tumor evolution, neural somatic mosaicism and embryonic development.
线粒体拥有其自身的小型环状基因组的多个副本，该基因组已被广泛研究，以追踪现代真核细胞的进化，并发现与遗传疾病相关的重要突变。全基因组和外显子组测序使人们能够以单核苷酸分辨率在大量样本和实验条件下研究 mtDNA，从而能够解析遗传突变与表型之间的关系，并鉴定经典线粒体疾病以及慢性疾病、衰老和癌症中的获得性 mtDNA 突变。通过应用基于我们的 MToolBox 软件的计算管道，我们使用通过不同扩增方法（eWGA、DOP-PCR、MALBAC、MDA）获得的全基因组和外显子组测序数据以及来自具有高通量测序的转座酶可及染色质的单细胞分析 (scATAC-seq) 数据，在单细胞中重建 mtDNA 基因组，其中 mtDNA 序列预计是该技术的副产品。我们表明，除了通过 MALBAC 和 DOP-PCR 方法重建的 mtDNA 之外，组装的 mtDNA 相当均匀，适合基因组研究，能够研究与细胞异质性相关的各种生物学过程，如肿瘤进化、神经体体细胞镶嵌和胚胎发育。
