Abstract 4244: Detection and characterization of extrachromosomal circular DNA in human plasma
Authors: Zhu Jing , Du Meijun , Zhang Peng , Wang Liang 
Published: July 2017

Abstract
Background: eccDNA (extrachromosomal circular DNA) has been reported in most eukaryotes, including in human cells. However, little is known about the eccDNA profiles in human circulating system such as blood.
Method: We extracted plasma cell free DNA (cfDNA) from three patients with advanced stage cancers. For each patient, we tested 10ng, 1ng and 0.1ng cfDNA, each with a technical replicate. The cfDNA samples were subjected to ATP-dependent DNase digestion and whole genome amplification (WGA). An additional 2ng cfDNA from each patient skipped the DNase digestion and went directly to WGA step. We sequenced the amplified DNAs using the Illumina 100bp PE read protocol. We analyzed sequencing data using a custom split reads-based strategy to retrieve circular DNA molecules. We further analyzed eccDNAs for their biofeatures using functional annotation tools.
Results: We received sequence reads at ~14.8 million (11.4~22.4) for each sample. When read depth≥ 3 as a cutoff, we detected a total number of 2542, 1578, and 57 unique eccDNAs in three patients while negative controls showed &lt;=5 eccDNAs. For non-DNase digested cfDNAs, we detected only 3 eccDNAs. By comparing read numbers mapped to mitochondria and human genome, we found that mitochondrial sequence reads increased significantly from 0.34-1.1% before digestion to 24.1-50.1% after digestion. Numbers of detected eccDNAs were similar between technical replicates and proportional to initial input cfDNA. However, eccDNAs detected also showed few overlaps between technical replicates. Size distribution ranged from 31bp to 19740bp (median 1801-1962bp). To test if eccDNAs were generated in specific genomic regions and if eccDNA-involved genes were enriched in specific pathways, we performed enrichment analysis and observed higher GC content in smaller eccDNAs (&lt;500 bp, 48%) than larger ones (&gt;500bp, 41%) (p&lt;0.001). When compared to random distribution, eccDNAs were more likely located at CpG islands (1.36-1.53 fold enrichment). The eccDNAs were also enriched in exons, 5’UTR, 3’UTR and DNase hypersensitive sites (enrichment folds from 1.39 to 2.3). Using DAVID annotation tool, we observed significant enrichment of the eccDNA-related genes in several categories including the Coiled coil, phosphoprotein, fibronectin, suggesting a potential involvement in gene expression regulation and cell adhesion events. Using GREAT (Genomic Regions Enrichment of Annotations Tool), we found that the eccDNAs were involved in genes associated with invasive cancer cell gene expression regulation (p&lt;0.001).
Conclusion: Human plasma contains an abundant number of eccDNAs. Distribution of these eccDNAs is nonrandom event and is likely enriched in genomic regions with known functional consequences and genes that play a role in gene regulations. Further characterization of circulating eccDNAs in peripheral blood will facilitate understanding of their molecular mechanisms and potential clinical utilities.
Citation Format: Jing Zhu, Meijun Du, Peng Zhang, Liang Wang. Detection and characterization of extrachromosomal circular DNA in human plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2017-4244