ABSTRACT
Circulating cell-free DNAs (cfDNAs) are DNA fragments which can be isolated from mammalian blood serum or plasma. In order to gain deeper insight into their origin(s), we have characterized the composition of human and cattle cfDNA via large-scale analyses of high-throughput sequencing data. We observed significant differences between the composition of cfDNA in serum/plasma and the corresponding DNA sequence composition of the human genome. Retrotransposable elements and non-telomeric satellite DNA were particularly overrepresented in the cfDNA population, while telomeric satellite DNA was underrepresented. This was consistently observed for human plasma, bovine serum and for the supernatant of human cancer cell cultures. Our results suggest that reverse transcription of retrotransposable elements and secondary-structure formation during the replication of satellite DNA are contributing to the composition of the cfDNA molecules in the mammalian blood stream. We believe that our work is an important step towards the understanding of the biogenesis of cfDNAs and thus may also facilitate the future exploitation of their diagnostic potential.
Subject(s)
Cell-Free Nucleic Acids/genetics , DNA, Satellite/genetics , Retroelements/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Cattle , Cell-Free Nucleic Acids/blood , Exosomes/genetics , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Nucleic Acid Conformation , Sequence Analysis, DNA , Young AdultABSTRACT
We have identified 24 molecular markers, based on circulating nucleic acids (CNA) originating from the human genome, which in combination can be used in a quantitative real-time PCR (qPCR) assay to identify the presence of human sepsis, starting two to three days before the first clinical signs develop and including patients who meet the SEPSIS-3 criteria. The accuracy was more than 87 % inside of the same patient cohort for which the markers were developed and up to 81 % in blind studies of patient cohorts which were not included in the marker development. As our markers are host-based, they can be used to capture bacterial as well as fungal sepsis, unlike the current PCR-based tests, which require species-specific primer sets for each organism causing human sepsis. Our assay directly uses an aliquot of cell-free blood as the substrate for the PCR reaction, thus allowing to obtain the diagnostic results in three to four hours after the collection of the blood samples.
