Cell-Free DNA Comprises the Strand-Specific Characteristic Associated with Transcription and Methylation

Abstract Background Cell-free DNA (cfDNA) is a promising biomarker and carries information about physiological and pathological states. Nucleosome and transcription factor footprints have been extensively studied based on double-stranded cfDNA sequencing; however, protein–DNA interaction footprints in single-stranded cfDNA fragments remain undercharacterized. Methods We used a single-stranded library preparation and sequencing method to investigate a new characteristic of cfDNA—strand specificity, based on the depth differences between the 2 DNA strands. The plasma cfDNA of 34 healthy individuals was analyzed to evaluate strand-specific bias on chromosomes and the mitochondrial genome. Then, the cfDNA samples were compared between 17 males and 17 females, as well as between 14 lung cancer patients and 18 noncancer controls. Results Short cfDNA (<160 bases) had higher strand-specific bias than long fragments. CfDNA strand-specific-biased regions were more enriched in chromosomes with higher gene densities and enriched in promoters, exons, 5′ untranslated region (UTR), 3′ UTR, and CpG islands. Genomic regions with higher CpG densities and lower methylation levels were more likely to generate strand-specific bias. For the mitochondrial DNA (mtDNA), the cfDNA strand-specific bias was pronounced in the noncoding region, exhibiting the footprints of mtDNA replication and transcription. Significant molecular sex differences could be revealed on cfDNA at the gene level, and distinguished features of lung cancer patients were also discovered. Conclusions As a proof-of-concept, we demonstrated the existence of a cfDNA strand-specific characteristic, which reflects footprint signatures including transcription and methylation. This study provides insights into the biological properties of cfDNA and a potential novel role as biomarkers.