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

Abstract

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 L1MA 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.

Keywords::

• apoptosis
• circulating DNA
• next-generation sequencing
• SOLiD platform