Massively Parallel Sequencing of Cell-free DNA in Maternal Plasma: Biological Profiling and Clinical Applications

Cell-free fetal DNA molecules exist as a minor population in a sea of maternally-derived DNA molecules in maternal plasma. This discovery has revolutionized prenatal genetic testing by providing a noninvasive source of fetal genetic material. The recent advent in massively parallel sequencing technologies has allowed precise quantitative analysis of fetal DNA in maternal plasma that has led to the development of several sequencingbased approaches for the prenatal detection of fetal aneuploidies and monogenic diseases. In addition, the use of paired-end sequencing has allowed absolute sizing of millions of plasma DNA molecules at single-base resolution through which has revealed distinctive fragmentation patterns of fetal and maternal DNA. In this thesis, novel uses of massively parallel sequencing were developed for the analysis of circulating fetal DNA in maternal plasma. The studies that have been addressed include the development of a protocol for determining the fetal karyotype noninvasively; the development of an approach based on size analysis of plasma DNA for the prenatal detection of fetal chromosomal aneuploidies; and an investigation of the clearance profile of circulating fetal DNA in a high resolution manner.;In the first part of this thesis, I have demonstrated the feasibility of performing prenatal detection of fetal subchromosomal copy number aberrations at 3-Mb resolution across the whole genome. Using an optimized diagnostic algorithm, fetus-derived subchromosomal deletions or duplications were successfully detected in five cases. In one case, maternally-derived microduplication was detected. The diagnostic sensitivity of prenatal fetal karyotyping was further explored using simulation analyses.;In the second part of this thesis, I have demonstrated the feasibility of prenatal detection of fetal aneuploidy by a size-based approach using paired-end massively parallel sequencing. By detecting a perturbed size distribution in an aneuploid chromosome, fetal chromosomal aneuploidies, including trisomy 21, 18, 13 and monosomy X, were successfully detected. In particular, 100% sensitivity and specificity were achieved for the detection of trisomies 21 and 18. This size profiling method could also improve the specificity of genomewide fetal aneuploidy detection when used synergistically with the previously developed tag counting method.;The last part of this thesis depicts the clearance profile of circulating fetal DNA in a high resolution manner. Serial maternal plasma and urine DNA samples collected from women after cesarean delivery were analyzed with paired-end massively parallel sequencing. The final disappearance and the clearance kinetics of circulating fetal DNA were studied. In addition, the contribution of transrenal excretion as a route for the clearance of circulating fetal DNA was investigated. This study not only unveils a biphasic clearance pattern of circulating fetal DNA, but also provides some mechanistic insights into the release and clearance of DNA in the maternal circulation.;In conclusion, the work presented in this thesis has provided a high-resolution clearance profile of circulating fetal DNA, further expanded the diagnostic spectrum of noninvasive prenatal tests, and opened up a new dimension for plasma DNA-based molecular diagnosis.