Global characterization of extrachromosomal circular DNAs in four body fluids

Determination of the type of body fluids is highly desired in forensic investigations. Extrachromosomal circular DNA (eccDNA) is a double-stranded, circular type of nucleic acid molecule that exists outside chromosomes and is ubiquitous in eukaryotic cells, which is widely explored in clinical research. The use of eccDNA for body fluid identification has remained relatively unexplored. As such, peripheral blood, menstrual blood, semen, and saliva samples (each = 3) from healthy individuals were detected, using Circle-seq, to identify potential markers for body fluid identification. In total, 263,036 eccDNAs were identified, widely distributed throughout the genome, and most of them were within 1000 base pairs in size. The study combined eccDNA with RNA analysis in an attempt to observe the relationship between eccDNA and gene expression in healthy individuals. We performed outward PCR on selected eccDNAs using library samples and validated the predicted junction sites by Sanger sequencing. In short, this pilot study demonstrated for the first time the characterization of eccDNA profiles from four body fluids of healthy individuals. It provided a novel direction for body fluid identification. In the future, breaking the low abundance and high heterogeneity of eccDNA will lead to new possibilities in the forensic field.