Extended Data Fig. 2: Sequencing saturation analysis and quality controls of MeDIP–seq and cfMeDIP–seq carried out on varying starting inputs of HCT116 DNA sheared to mimic cfDNA.
From: Sensitive tumour detection and classification using plasma cell-free DNA methylomes
a, Results of the saturation analysis from the Bioconductor package MEDIPS analysing cfMeDIP–seq data from each replicate, for each starting input amount and including an input control. b, The protocol was tested in two biological replicates of four starting DNA inputs (100, 10, 5 and 1 ng) of HCT116 DNA sheared to mimic cfDNA. The specificity of the reaction was calculated using methylated and unmethylated spiked-in A. thaliana DNA. The fold-enrichment ratio was calculated using genomic regions of the fragmented HCT116 DNA (human methylated HIST1H2BA and unmethylated GAPDH). The horizontal dotted line indicates a fold-enrichment ratio threshold of 25, dots represent biological replicates, with lines representing the mean. c, CpG enrichment scores of the sequenced samples (two biological replicates each of four starting DNA inputs (100, 10, 5 and 1 ng) and one input control) show a robust enrichment of CpGs within the genomic regions from the immunoprecipitated samples compared to the input control. The CpG enrichment score was obtained by dividing the relative frequency of CpGs of the regions by the relative frequency of CpGs of the human genome. The horizontal dotted line indicates a CpG enrichment score of 1, dots represent biological replicates, with lines representing the mean. d, Genome-wide Pearson correlations of normalized read counts per 300-bp window between cfMeDIP–seq signal for 1 to 100 ng of input HCT116 DNA sheared to mimic cfDNA (2 biological replicates per concentration). e, Genome Browser snapshot of HCT116 cfMeDIP–seq signal across a window (chr8:145,095,942–145,116,942) selected out of four examined loci, at different starting DNA inputs (1 to 100 ng, in biological replicates), compared with RRBS (ENCODE: ENCSR000DFS) and WGBS (Gene Expression Omnibus: GSM1465024) data (aligned to hg19). For cfMeDIP–seq, the y axis indicates RPKMs; for RRBS, yellow and blue blocks represent hypermethylated and hypomethylated CpGs, respectively. In the WGBS track, peak heights indicate methylation level.
