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A comparative study of cell-free apoptotic and genomic DNA using FISH and massive parallel sequencing

机译：利用FISH和大规模平行测序对无细胞凋亡和基因组DNA进行比较研究

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Objective: Study of circulating DNA (cirDNA) generation mechanisms with respect to their influence on the content of cirDNA is very important since it could indicate the best molecular targets for diagnostic applications. Since apoptosis was shown to be one of the main sources of cirDNA, we performed in vitro comparative study of cell-free apoptotic and genomic DNA (gDNA). Methods: DNA isolated from culture medium of apoptotic human umbilical vein endothelial cells (cm-apoDNA) and the gDNA from the same living cells was analyzed using FISH and sequenced on SOLiD 3 platform. Results/conclusions: FISH demonstrates overrepresentation of C-positive chromosome regions in cm-apoDNA. SOLiD 3 data show enrichment of cm-apoDNA for Alu repeats: the content of AluJ, AluS and AluY repeats was, respectively, 2.47-fold (standard deviation (SD) 3.6%), 2.45-fold (SD 5.5%) and 2.79-fold (SD 6.1%) higher in cm-apoDNA. By contrast, some of L1 elements were underrepresented in cm-apoDNA: the content of LIMA and L1ME was, respectively, 1.4-fold (SD 22%) and 1.45-fold (SD 9%) lower in cm-apoDNA. In contrast to FISH, these data and the predominant location of Alu repeats in euchromatic regions evidence the non-uniform gDNA degradation during apoptosis leading to the enrichment of cm-apoDNA with coding sequences.
机译：目的：研究循环DNA（cirDNA）的生成机制对其对cirDNA含量的影响非常重要，因为它可以为诊断应用提供最佳分子靶标。由于凋亡被证明是cirDNA的主要来源之一，因此我们进行了无细胞凋亡和基因组DNA（gDNA）的体外比较研究。方法：使用FISH技术分析从凋亡的人脐静脉内皮细胞（cm-apoDNA）培养基中分离的DNA和来自同一活细胞的gDNA，并在SOLiD 3平台上测序。结果/结论：FISH证明了cm-apoDNA中C阳性染色体区域的过度表达。 SOLiD 3数据显示cm-apoDNA对于Alu重复序列富集：AluJ，AluS和AluY重复序列的含量分别为2.47倍（标准偏差（SD）3.6％），2.45倍（SD 5.5％）和2.79- cm-apoDNA的折叠倍数（SD 6.1％）。相比之下，cm-apoDNA中的某些L1元素含量不足：LIMA和L1ME的含量分别比cm-apoDNA低1.4倍（SD 22％）和1.45倍（SD 9％）。与FISH相比，这些数据和Alu重复在常染色体区域中的主要位置证明了凋亡期间导致g-apoDNA富集编码序列的gDNA降解不均匀。

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