Abstract
BackgroundCirculating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors.ResultsWe perform whole genome bisulfite sequencing on a set of unmatched samples including circulating cell-free DNA from non-pregnant and pregnant female donors and genomic DNA from maternal buffy coat and placenta samples. We find CpG cytosines within longer fragments are more likely to be methylated. Comparison of the methylomes of placenta and non-pregnant circulating cell-free DNA reveal many of the 51,259 identified differentially methylated regions are located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases. We find these placenta hypomethylated domains are consistently located within regions exhibiting low CpG and gene density. Differentially methylated regions identified when comparing placenta to non-pregnant circulating cell-free DNA are recapitulated in pregnant circulating cell-free DNA, confirming the ability to detect differential methylation in circulating cell-free DNA mixtures.ConclusionsWe generate methylome maps for four sample types at single-base resolution, identify a link between DNA methylation and fragment length in circulating cell-free DNA, identify differentially methylated regions between sample groups, and uncover the presence of megabase-size placenta hypomethylated domains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0645-x) contains supplementary material, which is available to authorized users.
Highlights
Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA
Single base resolution methylome maps of ccf DNA isolated from the plasma of eight non-pregnant female donors were produced using whole genome bisulfite sequencing (WGBS)
This led us to perform WGBS on buffy coat cells obtained from seven distinct pregnant female donors (Additional file 2: Figure S2)
Summary
The field of non-invasive prenatal testing was enabled by the discovery that circulating cell free (ccf ) fetal DNA is present pregnant female plasma [1] It does, only present the minority species of DNA in total ccf DNA obtained from pregnant women. This mixture consists of DNA inherently present in the plasma of non-pregnant females, thought to primarily be derived from maternal hematopoietic cells, supplemented with a minority fraction of fetal DNA coming from the placenta [2,3,4,5] Since these nucleic acids are distinct, they can be differentiated through a number of genomic markers including single nucleotide changes, haplotypes, or copy number variants. Utilizing sequencing techniques that permit complete methylome analysis, a number of studies have described genome-wide methylation profiles of normal and cancer samples [22,23,24,25,26,27,28,29,30]; high resolution methylation maps of complex biological specimens including ccf DNA only recently been described [31]
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