show Abstracthide AbstractThree separate experiments were carried out using MeDIP-seq and cfMeDIP-seq for methylome analysis. For the first experiment, different starting amounts of HCT116 cell line DNA, sheared to mimic cell-free DNA, were analyzed using MeDIP-seq and cfMeDIP-seq. In the second experiment the limit of detection of cfMeDIP-seq was tested using varying dilutions of colorectal cancer cell line DNA (HCT116) with multiple myeloma cell line DNA (MM1.S). For both cell line DNA samples, the DNA was sheared to mimic cell-free DNA. In the final experiment, we tested the enrichment of human ctDNA using cfMeDIP-seq performed on plasma collected from patient-derived xenografts (PDXs) generated in mice from two colorectal cancer patients. Overall design: The gold standard MeDIP-seq protocol using 100 ng of sheared cell line DNA and the cfMeDIP-seq protocol using 10 ng, 5 ng, and 1 ng of the same sheared cell line DNA was performed with two biological replicates. For the samples starting with less than 100 ng of DNA, as part of cfMeDIP-seq, the DNA starting amount prior to MeDIP was inflated to 100 ng using filler DNA in the form of unmethylated and artificially methylated PCR amplicons. For the second experiment, using HCT116 and MM1.S cell line DNA sheared to mimic cell-free DNA, a serial dilution was performed using a starting DNA amount of 60 ng with HCT116 DNA diluted with MM1.S DNA. The varying percentages of HCT116:MM1.S DNA included: 100% HCT116, 10% HCT116, 1% HCT116, 0.1% HCT116, 0.01% HCT116, 0.001% HCT116 and 100% MM1.S. These samples were subjected to cfMEDIP-seq, with the DNA starting amounts prior to MeDIP inflated to 100 ng using filler DNA as previously mentioned. In both experiment 1 and 2, the sequenced reads were aligned to reference genome hg19. For the PDX-driven experiment, cfMeDIP-seq was performed on two plasma samples from mice engrafted with colorectal cancer patients-derived xenograft (PDXs). In this experiment, both the cfMeDIP-seq samples and their respective input controls were sequenced. The sequencing reads were aligned to both reference genome hg19, and reference genome mm9. The PDX samples (GSM2422520 - GSM2422527) have no associated processed data. Conclusions for these samples were based solely on alignments. Raw data for these samples are represented by BAM files.