Same-day genomic and epigenomic analysis of brain tumours case study

A plethora of technologies are currently required to assess different genomic and epigenomic alterations; however, the associated costs and long turnaround times combined with extensive infrastructure and training requirements have, to date, hindered their implementation¹ . To address these challenges, Dr. Philippe Euskirchen and co-workers at the ICM Brain and Spine Institute, France, assessed the potential of nanopore sequencing technology to deliver comprehensive and cost-effective characterisation of genetic alterations in brain cancer samples — including analysis of copy number (CN) alterations, epigenetic base modifications, and single nucleotide variations (SNVs)^1,2. Furthermore, all nanopore sequencing workflows were designed to go from sample to result within a single day.

‘Methylation data can directly be obtained from the same WGS data set which makes time-consuming bisulfite conversion and specialized methylation assays (sequencing or hybridizationbased) expendable’¹.

Using a low-pass whole genome sequencing approach, the team analysed both CN alterations and base modifications in a number of previously characterised brain tumour samples. The six-hour, real-time nanopore sequencing runs, which delivered <0.01x to 0.24x genome coverage, allowed detection of chromosome arm-level CN alterations and epigenetic profiles with high concordance to matched microarray data (Figures 1 and 2). The low-pass methylation data allowed reliable subtyping of glioma samples into IDH-mutant and wild type. Mutations in the IDH genes typically result in global hypermethylation of CpG islands, which is associated with favourable prognostic outcomes. Significantly, the nanopore sequencing results were obtained within hours, as opposed to approximately 2 weeks as required for microarray-based analysis¹ .

Next, the team demonstrated the potential of the CN and methylation data to enable the identification of specific cancer types. All seven glioma samples tested were correctly identified using this approach. In addition, the team were able to determine the origin of several brain metastasis samples, including one instance of a breast adenocarcinoma metastasis found in the posterior fossa, for which immunohistochemistry had provided misleading results.

Figure 1: Comparison of copy number profiles obtained using lowpass whole genome nanopore sequencing and SNP arrays for matched samples. Image adapted from Euskirchen et al1 and available under Creative Commons license (creativecommons.org/ licenses/by/4.0).

Figure 2: Comparison of methylation calls from nanopore sequencing with matched Illumina 450K microarray data. Beta value distributions for CpG sites that were identified as unmethylated (red) or methylated (blue), respectively, by nanopore WGS are shown. Image adapted from Euskirchen et al¹ and available under creative commons license (creativecommons.org/licenses/by/4.0).

To detect known SNVs, the researchers designed an amplicon panel covering hotspot exons in IDH1, IDH2, and H3F3A, all coding exons of TP53 and, additionally, the TERT promoter (pTERT) region. Due to the long reads delivered by nanopore sequencing, this could be achieved with just nine PCR reactions. Using the real-time data acquisition capabilities of nanopore sequencing, the researchers were able to stop sequencing once the desired read depth of 1,000x was reached for all amplicons, which was achieved in a timeframe of just 2–20 minutes. In all samples, coding mutations were reliably detected as compared to routine analysis based on Sanger sequencing, immunohistochemistry, or a next generation sequencing (NGS) panel.

Furthermore, the facility for sample multiplexing (WGS – 4 samples; amplicon – 12 samples) provided highly cost-effective analyses. Confirming the advantaged of nanopore sequencing for cancer research, the team commented, ‘Nanopore sequencing allows same-day detection of structural variants, point mutations, and methylation profiling using a single device with negligible capital cost’¹

This case study was taken from the clinical white paper.