Comprehensive analysis of the tissue of origin of plasma cell-free DNA (cfDNA) remains insufficient. A genome-scale DNA methylation method for this analysis is of both biological and clinical interest.

We used the methylated CpG tandem amplification and sequencing (MCTA-Seq), which is a genome-scale DNA methylation method, for analyzing cfDNA. We performed MCTA-Seq to pair plasma cfDNA and white blood cell genomic DNA from 14 healthy individuals for comparative analysis, with eight tissues being analyzed for identifying tissue-specific markers. The relative contributions of multiple tissues to cfDNA were calculated for plasma cfDNA obtained from healthy adults (n = 25), cholelithiasis patients (n = 13), liver cirrhosis patients (n = 17), hepatocellular carcinoma patients (n = 30), and acute pancreatitis patients (n = 8).

We identified a total of 146 tissue-specific hypermethylation markers. Simulation analysis showed that MCTA-Seq can accurately measure DNA fractions contributed by multiple tissues to cfDNA. We demonstrated that the liver is the major non-hematopoietic tissue contributing to plasma cfDNA in healthy adults. The method also detected increases in the liver-derived DNA in the blood from patients with liver diseases, which correlate with an increase in the liver enzyme level. Furthermore, the results indicated that blood cells make a major contribution to the elevation of cfDNA levels in acute pancreatitis, liver cirrhosis, and hepatocellular carcinoma patients. Finally, we characterized a novel set of tissue-specific hypermethylation markers for cfDNA detection, which are located within the intragenic regions of tissue-specific highly expressed genes.

We have used MCTA-Seq for simultaneously measuring cfDNA fractions contributed by multiple tissues. Applying this approach to healthy adults and liver and pancreas disease patients revealed the tissue of origin of cfDNA. The approach and the identified markers should facilitate assessing the cfDNA dynamics in a variety of human diseases.