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.