WGS of Cell free DNA : Homo sapiens
Briefly, archival frozen plasma specimens stored at 80 C were thawed and centrifuged at 16,000g (5 minutes) x3 to remove the cryoprecipitates. cfDNA was isolated using the QIAamp Circulating Nucleic Acid Kit (QIAGEN Cat. no. 55114). 1.3ng/L carrier RNA was used to improve yield. cfDNA fragment size distribution and concentration were respectively measured using Agilent TapeStation 4200 HS D1000 tape cartridges and Qubit dsDNA high sensitivity assay (ThermoFisher Invitrogen Cat. no. Q32851) Plasma DNAs 0.1ng total with size distributions <1000bp were used to construct sequencing libraries with Hyper Prep Kit (Roche KAPA Cat. no. KK8504) as per the manufacturers instructions. After ligation, the cfDNA sequencing libraries were cleaned with 0.8X AMPure XP Beads (Beckman Coulter Cat. no. A63881). Reference normal genomic DNA was isolated from PBMC with QIAamp DNA Micro Kit (QIAGEN Cat. no. 56304), as per the manufacturers instructions. The isolated DNA quality was measured using TapeStation genomic DNA tapes and the concentration measured using Qubit dsDNA high sensitivity assay. Genomic DNA was sonicated to 200bp (Covaris Peak power 175, Duty Factor 10%, cycles/burst 200, time 180s, temp 47C) and the libraries were constructed using Hyper Prep Kits, following the manufacturers instructions. The reference gDNA libraries were cleaned with 0.8X AMPure XP Beads.
SUB14805063
PR126T1