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标题：Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma
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作者：Stephanie C. Y. Yu ; Peiyong Jiang ; Wenlei Peng 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：50
DOI：10.1073/pnas.2114937118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance We revealed a large population of long cell-free DNA molecules (up to 23,635 bp in length) in maternal plasma and developed an approach which leveraged the abundance of CpG sites on long molecules to deduce the tissue of origin of individual plasma DNA molecules based on single-molecule methylation analysis. We illustrated how such an approach may be utilized to achieve noninvasive prenatal testing of monogenic diseases. We also revealed a reduction in amounts of such long cell-free DNA molecules and a different end motif profile in maternal plasma DNA from pregnancies with preeclampsia. Hence, long cell-free DNA molecules represent a valuable resource of biomarker development for pregnancy-associated disorders. In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5′ fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5′ 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.
关键词：cell-free DNA; noninvasive prenatal testing; third-generation sequencing; epigenetics; monogenic diseases