Liver transplant cell-free DNA sample at FC-POD18
Circulating cell-free DNA was extracted from 4-6 mL human serum, using the QIAamp Circulating Nucleic Acid kit (Qiagen, Cat. No. 55114) according to the manufacturer's instructions. Cell-free DNA was quantified via Qubit fluorometer using the dsDNA Assay Qubit fluorometer Kits (ThermoFisher Scientific, Cat. No. Q32853 and Q10212 respectively). As a quality control, fragment size distribution of isolated cfDNA was verified based on analysis using a 2100 Bioanalyzer TapeStation using either a High Sensitivity DNA Assay or DNA 1000 Assay (Agilent Technologies, Santa Cruz, CA, USA) dependent on concentration determined via either Qubit dsDNA BR or HS Assay. Additional purification using Beckman Coulter beads was implemented to remove high-molecular weight DNA reflective of cell-lysis and leukocyte contamination (Agencourt AMPure cat#A63880) (Maggi et al. 2018). Whole Genome Bisulfite Sequencing libraries were generated using the Zymo Research Pico Methyl-Seq Library Prep Kit (#D5455 Zymo Research, Irvine, CA). WGBS libraries were quantified using KAPA Library Quantification Kits for Illumina Platforms (Kapa Biosystems; Wilmington, MA) and then pooled to meet the required 1ug DNA input necessary for targeted enrichment. Hybridization capture was carried out according to the SeqCap Epi Enrichment System protocol (Roche NimbleGen, Inc.; Pleasanton, CA) using xGen Universal Blocker-TS Mix (Integrated DNA Technologies, USA) as the blocking reagent and SeqCap Epi CpGiant probe pools. MethylC-Capture Sequencing (MCC-seq) libraries were assessed using Qubit Fluorometer (ThermoFisher) and Agilent Bioanalyzer DNA 1000 assays (Agilent Technologies, Inc., USA). MCC-seq libraries with inclusion of 15-20% spike-in PhiX Control v3 library (Illumina; San Diego, CA) were clustered on an Illumina Novaseq 6000 S4 flow cell followed by 150-bp paired-end sequencing.