Whole Genome Bisulfite Sequencing of ccf DNA and its Cellular Contributors

Background: Circulating cell free (ccf) fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the genetically distinct maternal and fetal DNA. Current testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA and thus support additional clinical opportunities; however, this depends on knowledge of the methylomes of ccf DNA and its cellular contributors. Results: Whole genome bisulfite sequencing was performed on a set of unmatched samples including ccf DNA from 8 non-pregnant (NP) and 7 pregnant female donors and genomic DNA from 7 buffy coat and 5 placenta samples. We found CpG cytosines within longer fragments were more likely to be methylated, linking DNA methylation and fragment size in ccf DNA. Comparison of the methylomes of placenta and NP ccf DNA revealed many of the 51,259 identified differentially methylated regions (DMRs) were located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases, regions we termed placenta hypomethylated domains. DMRs identified when comparing placenta to NP ccf DNA were recapitulated in pregnant ccf DNA, confirming the ability to detect differential methylation in ccf DNA mixtures. Conclusions: We generated methylome maps for four sample types at single base resolution, identified a link between DNA methylation and fragment length in ccf DNA, identified DMRs between sample groups, and uncovered the presence of megabase-size placenta hypomethylated domains. Furthermore, we anticipate these results to provide a foundation to which future studies using discriminatory DNA methylation may be compared.]]>
Specimens analyzed for this study were collected under three separate IRB approved studies. Plasma was extracted from whole blood specimens from pregnant subjects collected under protocol SQNM-T21-106 (Compass IRB 00462). Subjects provided written informed consent and were 18 years of age or older and pregnant carrying a singleton fetus of 10 to 26 weeks gestational age inclusive. Plasma was extracted from whole blood specimens from non-pregnant female subjects collected under protocol SQNM-RND-103 (Aspire IRB T21106). Subjects provided written informed consent and were normal healthy volunteers 18 years of age or older confirmed not to be pregnant. Peripheral blood mononucleated cells (PBMCs or buffy coat) were extracted from whole blood specimens from pregnant subjects collected under protocol 1128724 (Western IRB 20111833). Placental tissue was collected from subjects under the same protocol (1128724). Subjects provided written informed consent and donated the whole blood and aborted material as an anatomical gift for the purposes of education, research, or for the advancement of medical science.]]>
Whole genome bisulfite sequencing was performed on a set of 27 unpaired samples from four distinct groups: five placenta villi gDNA samples, seven maternal buffy coat gDNA samples, eight ccf DNA samples from the plasma of non-pregnant females, and seven ccf DNA samples from the plasma of pregnant female donors. Differential methylation was confirmed using EpiTYPER analysis in an independent sample cohort.]]>