<p>We performed a pilot study in which Illumina (ILMN) and nanopore (ONT) whole genome sequencing (WGS) was generated for samples obtained from a 37-year-old patient with locally advanced rectal cancer who did not respond to neoadjuvant chemoradiotherapy (nCRT). Pre-treatment and post-treatment tumour biopsy, adjacent normal tissue, and matched pre-treatment blood samples were sequenced to identify somatic alterations. We used DNA methylation from ONT WGS to detect changes in the tumour epigenomes compared to adjacent normal tissue. We employed established pipelines for the identification of somatic alterations to assess the concordance of SNV, short indel, copy number and structural variation from the short-read and long-read tumour genome data. Overall, 69.4% and 30.1% of SNVs were concordant between technologies in the pre-and post-treatment tumours, respectively. A multinomial logistic regression was performed to further understand discordant SNV calls, highlighting tumour purity, presence in a repetitive region, strand bias, and germline variant allele frequency as factors contributing to discordance. Indel concordance was poor (26.8% and 9.2% in the pre-and post-treatment tumour, respectively). Copy number alteration profiles were highly similar while minimal concordance was reported for structural variants (2.4% and 0.3% for pre-and post-treatment tumours, respectively). Finally, we used the ONT data to detect DNA modifications (5-methylcytosine and 5-hydroxymethylcytosine) between tumour and matched adjacent normal tissue. Based on genome-wide average CpG modification rates, global hypomethylation was observed in tumours compared to adjacent normal tissues. Biologically relevant epigenomic changes related to epithelial-mesenchymal transition were detected in the post-treatment tumour, as well as potential radiation-induced changes in the post-treatment adjacent normal tissue.</p>