Ultra-low DNA input into whole genome methylation assays and detection of oncogenic methylation & copy number variants in circulating tumour DNA

BackgroundCpG methylation in cancer is ubiquitous and generally detected in tumour specimens using a variety of techniques at a resolution encompassing single CpG loci to genome wide. Analysis of samples with very low DNA inputs, such as formalin fixed (FFPE) biopsy specimens from clinical trials or circulating tumour DNA has been challenging and has only been typically at single CpG sites. Analysis of genome wide methylation in these specimens has been limited because of the relative expense of techniques need to carry this out. We present the results of low input experiments into the Illumina Infinium HD methylation assay on FFPE specimens and ctDNA samples. MethodsFor all experiments, the Infinium HD assay for Methylation was used. In total, forty-eight FFPE specimens were used at varying concentrations (lowest input 50ng), eighteen blood derived specimens (lowest input 10ng) and six matched ctDNA input (lowest input 10ng) / fresh tumour specimens (lowest input 250ng) were processed. Downstream analysis was performed in R/Bioconductor for QC metrics and differential methylation analysis as well as copy number calls. ResultsCorrelation coefficients for CpG methylation at the probe level averaged R2=0.99 for blood derived samples and R2>0.96 for the FFPE samples. When matched ctDNA/fresh tumour samples were compared R2>0.91. Results of differential methylation analysis did not vary significantly by DNA input in either the blood or FFPE groups. There were differences seen in the ctDNA group as compared to their paired tumour sample, possibly because of enrichment for tumour material without contaminating normal. Copy number variants observed in the tumour were generally also seen in the paired ctDNA sample. ConclusionsThe Illumina Infinium HD methylation assay can robustly detect methylation across a range of sample types, including ctDNA, down to a input of 10ng. It can also reliably detect oncogenic methylation changes and copy number variants in ctDNA.