Abstract
BackgroundHigh-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data.MethodsIn this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome.ResultsWe show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA.ConclusionsThese background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection. Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0107-z) contains supplementary material, which is available to authorized users.
Highlights
High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA
We show that the mapping locations of cell-free DNA fragments associate with arrays of nucleosomes on a genome-wide level by correlating them with nucleosome and open chromatin enrichment positions from The Encyclopedia of DNA Elements (ENCODE)
We examine the periodicity in cell-free DNA fragment length distribution using Fourier analysis and confirm the ~180 and ~10 bp periodicity due to the two levels of cleavage which others have only assessed visually
Summary
High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. Current understanding of cell-free DNA encompasses that it exists as double-stranded molecules, which are biologically fragmented into both short (10 Kb) [2, 3] This disparity in size along with evidence from experimental and observational studies have led researchers to postulate apoptosis [4, 5], necrosis [2, 6] and active release [4, 7] as potential mechanisms that may produce extracellular DNA. While a majority of cell-free DNA circulates as histone bound nucleosomal elements [13, 14], at least a portion of this DNA appears to be housed with lipoprotein virtosomes or held within membranous vesicles These are believed to grant protection against further enzymatic degradation and recognition by immune cells that could trigger autoimmune responses. Detailed information of the mechanisms associated with these processes is lacking and remain somewhat controversial
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