Library construction for NOIR-SS was performed according to a modification of a previously described procedure . For each plasma sample, we prepared two separate reaction mixtures with two gene-specific primers (shown with a “1” or “2” suffix in S2 Table) since our PCR system did not allow the use of primer pairs. Cell-free DNA obtained from approximately 1 ml of whole blood (average 4.5 ng, median 3.1 ng).was end-repaired in a 15 μL reaction containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, 0.4 mM dNTPs, 2.4 units of T4 DNA polymerase (Takara Bio, Kusatu, Japan), 7.5 units of T4 polynucleotide kinase (NEB, Ipswich, MA, USA), and 0.5 units of KOD DNA polymerase (Toyobo, Osaka, Japan), which was incubated for 30 minutes at 25°C and then for 20 minutes at 75°C. Adapters tagged with the 12-nucleotide barcode sequence were ligated in a 20 μL end-repair reaction containing 0.5 μL of 10× T4 DNA ligase buffer (NEB), 40 pmol of adapter, and 2000 units of T4 DNA ligase (NEB), which was incubated at 25°C for 15 minutes. The ligation products were purified twice with 1.2× volumes of AMPure XP beads (Beckman Coulter, Brea, CA, USA). The purification beads were then mixed with 20 μL of the linear amplification reaction mix (1× Q5 Reaction Buffer [NEB], 0.2 mM dNTPs, 6 μM gene-specific primer mix [suffix “1” or “2” in S2 Table], and 0.4 units of Q5 Hot Start High-Fidelity DNA Polymerase [NEB]). After the AMPure XP beads were removed, the amplification was performed in the reaction: denaturation at 98°C for 30 seconds, and then 15 cycles of 10 seconds at 98°C and 2 minutes at 65°C. Then, 1.2 μL of 100 μM T_PCR_A was added to the reaction, and the mixture was incubated as follows: 15 cycles of 10 seconds at 98°C, 30 seconds at 65°C, and 30 seconds at 72°C. The amplification products were purified once with 1.2× volumes of AMPure XP, and recovered in 20 μL of 0.1× TE. Three microliters of the purified products were added to two tubes containing PCR amplification solution (20 μL each): 1× High Fidelity PCR Buffer (Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mM dNTPs, 2 mM MgSO4, 0.5 μM T_PCR_A, 0.5 μM nested-primer-mix (suffix “L” or “H” in S2 Table), and 0.4 units of Platinum Taq DNA Polymerase, High Fidelity (Thermo Fisher Scientific). The thermal cycling program was as follows: 2 minutes of denaturation at 95°C followed by 25 (nested-primer-mix with suffix “H”) or 30 (nested-primer-mix with suffix “L”) cycles of 15 seconds at 95°C and 1 minute at 63°C. The amplification products were purified with 1.2× volumes of AMPure XP beads. The product concentration was determined using the Qubit dsDNA HS Assay Kit or Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific).