Plasma cfDNA extraction and sequencing
For each blood collection, five milliliters of peripheral blood was obtained from the participating woman in an ethylene diamine tetraacetic acid-anticoagulated (EDTA) tube. Plasma was separated within 8 hours following a two-step centrifugation protocol [14]. Briefly, blood was centrifugated at 1600 g for 10 minutes at 4°C, and then the supernatant was transferred to a new Eppendorf tube for second centrifugation at 16000 g for 10 minutes at 4°C. The supernatant was taken into a new tube and stored at -80°C for future use. All subsequent procedures, including cfDNA isolation, library construction, and sequencing, were performed at an ISO/IEC 17025 certified laboratory of BGI-Shenzhen, China. CfDNA was extracted from 200μL plasma using a Micro DNA Kit (Tiangen) following the manufacturer’s instructions. The extracted cfDNA was end-repaired and then ligated with adaptors for multiplex sequencing. The ligated products were subjected to 12 cycles of amplification using the Kapa HIFI hotstart ready master mix (Kapa Biosystems), followed by quantitation using the dsDNA HS Assay kit 2.0 (Invitrogen) by Qubit2.0 and Agilent High Sensitivity DNA Kit with a 2100 Bioanalyzer (Agilent Technologies). PCR products were then normalized and processed for circularization using MGIEasyTM DNA Library Prep Kit (MGI, China) following the manufacturer’s instructions [15]. Briefly, PCR products were heat-denatured at 95 °C for 3 minutes to make single strand DNA circles (ssDNA circles), which were then mixed with reagents of MGIEasyTM DNA Library Prep Kit and incubated at 37 °C for 30 minutes to complete the circularization. The resulting ssDNA circle was then used to generate DNA nanoballs (DNBs) by rolling circle amplification (RCA) [16]. After RCA and the formation of DNBs, the final product was measured by Qubit (Thermo Fisher) using the ssDNA HS Assay kit (Invitrogen), and loaded on a DNBSEQ-500 platform (MGI, China) for sequencing [17] following the manufacturer’s instructions. Sequencing was conducted with a strategy of single-end 35 base pairs. The data that support the findings of this study have been deposited into CNSA (CNGB Nucleotide Sequence Archive)of CNGBdb with accession number CNP0000992 (https://db.cngb.org/cnsa/ ) upon the request of accession code.