Plasma Cell-Free DNA Sequencing Protocol

1. Collect 5mL of peripheral blood using separate EDTA blood tubes.
2. Extract maternal plasma by a two-step centrifugation procedure within 8 h after blood collection. Briefly, centrifuge maternal blood initially at 1600×g for 10min at 4°C, and transfer 1ml of supernatant to a new 1.5ml Eppendorf tube for a second centrifugation at 16000×g for 10min at 4°C. Then, transfer most maternal plasma to a new 1.5 ml Eppendorf tube. Be careful not to remove any buffy coat or bottom debris when transferring maternal plasma.
3. Extract cfDNA from 300μL of maternal plasma using a Circulating DNA Isolation Kit following the manufacturer's instructions. Briefly, mix 20μL of Proteinase K and 25μL of Pure Particle G with 300μL of maternal plasma in a new 1.5ml Eppendorf tube. Thoroughly mix and add 550μL of Lysis Buffer. Briefly vortex and put the Eppendorf tube on a magnetic separation device for 15min at room temperature.
4. Carefully remove all supernatant and mix the remaining beads with 700 μL of Wash Buffer 1. Briefly vortex and place the Eppendorf tube on a magnetic separation device for 1 min.
5. Carefully remove all supernatant and mix the remaining beads with 700 μL of Wash Buffer 2 (make sure ethanol is added). Briefly vortex and place the Eppendorf tube on a magnetic separation device for 1 min.
6. Carefully remove all supernatant and repeat washing with 700 μL of Wash Buffer 2. Place the Eppendorf tube on a magnetic separation device to carefully remove all supernatant, and then leave at room temperature to dry for 10 min.
7. Add 50 μL of elution buffer, briefly vortex, and leave at room temperature for 5 min. Place the Eppendorf tube on a magnetic separation device, and remove supernatant to a new Eppendorf tube.
8. Determine the quantification of the extracted plasma cfDNA using a Qubit ds DNA HS Assay kit, and determine the DNA fragment size using an Agilent 2100 kit on a Bioanalyzer 2100 platform. Plasma cfDNA should have a fragment peak at 166–170 bp.
9. Construct the cfDNA library using a Cell-free DNA Library Prep Set following the manufacturer's instructions. Use 5 ng of cfDNA to perform end-repair and A-tailing. Adapters with specific barcodes are ligated to the cfDNA fragments, and then the ligation product is cleaned with the provided Purification Beads.
10. After cleanup, the DNA is amplified by 12 cycles of PCR and another round of purification following the kit's instructions. Quantify the PCR products with dsDNA HS Assay Kit. The required yield for PCR products is ≥2 ng/μL.
11. Heat-denature the PCR products at 95 °C for 3 min on a PCR thermocycler. Mix the denatured DNA with the Circularization Reaction Mixture, and incubate at 37 °C for 30 min to create single-stranded DNA circles.
12. Use 20 μL of circularized ssDNA product for sequencing following Highthrough-put Sequencing Set Instruction Manual.
13. Quantify 2 μL of DNBs using ssDNA Assay Kit and Qubit Fluorometer. A minimum concentration of 8 ng/μL is required.
14. Load each DNB preparation on a separate lane to be processed for 100 bp paired-end (PE) sequencing following the manufacturer's protocol. Other sequencing instruments can be used, but sequencing oligonucleotides and conditions will need to be modified.