Profile of the Circulating DNA in Apparently Healthy Individuals

Abstract

Background: Circulating nucleic acids (CNAs) have been shown to have diagnostic utility in human diseases. The aim of this study was to sequence and organize CNAs to document typical profiles of circulating DNA in apparently healthy individuals. Methods: Serum DNA from 51 apparently healthy humans was extracted, amplified, sequenced via pyrosequencing (454 Life Sciences/Roche Diagnostics), and categorized by (a) origin (human vs xenogeneic), (b) functionality (repeats, genes, coding or noncoding), and (c) chromosomal localization. CNA results were compared with genomic DNA controls (n = 4) that were subjected to the identical procedure. Results: We obtained 4.5 × 105 sequences (7.5 × 107 nucleotides), of which 87% were attributable to known database sequences. Of these sequences, 97% were genomic, and 3% were xenogeneic. CNAs and genomic DNA did not differ with respect to sequences attributable to repeats, genes, RNA, and protein-coding DNA sequences. CNA tended to have a higher proportion of short interspersed nuclear element sequences (P = 0.1), of which Alu sequences were significant (P < 0.01). CNAs had a significantly lower proportion of L1 and L2 long interspersed nuclear element sequences (P < 0.01). In addition, hepatitis B virus (HBV) genotype F sequences were found in an individual accidentally evaluated as a healthy control. Conclusions: Comparison of CNAs with genomic DNA suggests that nonspecific DNA release is not the sole origin for CNAs. The CNA profiling of healthy individuals we have described, together with the detailed biometric analysis, provides the basis for future studies of patients with specific diseases. Furthermore, the detection of previously unknown HBV infection suggests the capability of this method to uncover occult infections.

Next Generation Sequencing of Serum Circulating Nucleic Acids from Patients with Invasive Ductal Breast Cancer Reveals Differences to Healthy and Nonmalignant Controls

“…Based on recent reports, a bias introduced by random amplification methods including the WGA4 cannot be ruled out (21,45), in which the latter was shown to introduce the least bias (45). To avoid a significant influence on the comparative data, samples were prepared strictly the same way, two independent WGA4 reactions were pooled for each sample and breast cancer samples and normal controls were run together in mixed batches to exclude bias introduced by individual sequencing runs.…”

“…A detailed patient description is given in Supplementary Table S1. Sequence data from 28 apparently healthy female controls were previously generated and stored in a CNA database that was described recently (21). Additional 87 collected samples of patients and controls were analyzed as an independent noncancerous validation set and 31 samples collected from a patient with multiple myeloma and liver sarcoma served as a malignant control (Table 1).…”

“…The serum DNA was processed, sequenced, and analyzed as described previously (21). Briefly, extracted DNA was amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (WGA4, Sigma-Aldrich) in duplicates, which were pooled for subsequent sequencing.…”

“…Although probably not necessary for CNA, the shearing procedure as included in the WGA4 protocol was done for comparability with, e.g., genomic results. Previously, we have tested the WGA4 procedure using either unsheared or sheared genomic DNA and could not detect essential differences in sequencing results (21). Using the adapters that are attached during the whole genome amplification procedure, a samplespecific molecular barcode was attached to the amplicons by PCR.…”

Cell-Free DNA: An Upcoming Biomarker in Transplantation

“…Different mechanisms accounting for DNA release in the circulation have been postulated, including apoptosis (23)(24)(25)(26), necrosis (26), and active secretion (27)(28)(29). Observations in gender-mismatched bone marrow and solid organ transplantation models point to hematopoietic cells as primary source of cfDNA in the plasma, with nonhematopoietic cells accounting for a minority of the free circulating DNA (24,30,31).…”