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See how cancer research can benefit from accessible, targeted long nanopore reads which facilitate a comprehensive analysis of multiple variant types across genomics, transcriptomics, and epigenetics.
Translational cancer research often focuses on characterising specific oncogenic mutations. In recent years, long nanopore sequencing reads have gained popularity in this field due to their advantages in resolving complex structural variants (SVs), phasing, and assembling repetitive regions. However, there is growing evidence that other factors, such as associated epigenetic effects, can also be of high importance for cancer research.
Healthcare aims to help as many people as much as possible, but is commonly limited by time and funding. These restraints push cancer researchers to investigate potential future methods of obtaining more comprehensive insights, more cost effectively, and with shorter turnaround times. Around the world, researchers are taking up this challenge, and at London Calling 2022, some of these researchers shared their experiences and breakthroughs using Oxford Nanopore technology*.
The multi-tool of cancer research genetics
One such researcher is Abderaouf Hamza, from the Curie Institute in Paris, France. Abderaouf spoke about his group’s use of MinION™ Mk1B devices for cancer research. He described the benefits of having a genomics multi-tool like the MinION at his disposal.
Abderaouf and his group study adult and paediatric cancers, genetic predispositions, and rare tumours. To do so, they seek to analyse SVs, single-nucleotide variants (SNVs), copy number variants (CNVs), gene expression, and methylation changes in cancer, which traditionally have required several different techniques and platforms. However, Abderaouf stated, ‘with the MinION, we can collect a wide range of the data we need on a single device.’
‘The multimodality, Swiss army knife character of the nanopore sequencer interested us’
Discussing the MinION sequence data obtained, Abderaouf emphasised the benefits of adaptive sampling. During this technique, exclusive to nanopore technology, each read is mapped to a reference in real time while DNA is passing through a nanopore. If a DNA strand aligns to a target region, sequencing continues, but if not, the strand is rejected, resulting in a short, off-target read. For their research, they selected 360 target gene sequences plus 10 kb of flanking regions, which accumulated to ~1.5% of the human genome. In this way, Abderaouf explained, adaptive sampling drastically reduces the cost per sample, making long sequencing reads more accessible.
While adaptive sampling generates long targeted reads that allow for phasing and gene fusion prediction, Abderaouf could also use the shorter rejected reads for shallow whole-genome sequencing (WGS) analysis. The team seized this opportunity to study CNVs and conduct a broader methylation analysis across the entire genome, as methylation information can be called directly from PCR-free nanopore sequencing data.
‘With adaptive sampling, you get way more data with the same sample; one MinION per sample is enough to get all we need.’
To validate the performance of adaptive sampling, Abderaouf and his team compared it to Oxford Nanopore WGS and WGS from an alternative long-read platform. They found more significant differences between variant callers than between these technologies. Abderaouf stated that his group had not seen any significant bias from adaptive sampling.
Utilising long, PCR-free nanopore reads for comprehensive variant analysis
Another cancer researcher who has taken up the challenge of using their nanopore data to its full potential is Kieran O’Neill, from Canada’s Michael Smith Genome Science Centre at BC Cancer, Canada. At London Calling, Kieran presented how his group evaluated the future potential of long nanopore sequencing reads for personalised oncogenomics (POG)s.
The advantages of long reads are no new discovery for Kieran’s group. In 2019, Kieran explained that clinical research samples with hereditary susceptibility variants, called via short-read sequencing, were re-sequenced with Oxford Nanopore technology. In several of these samples, the long nanopore reads allowed for more accurate variant calling. In one example, a variant previously called as a severe inversion between two oncogenes was revealed to be a translocation of an Alu element from one intron to another. Kieran stated that ‘this was completely invisible to short reads, but nanopore allowed us to see that it was actually a false positive.’
Kieran and his colleagues have more recently used their PromethION™ device to re-sequence samples with known breast cancer susceptibility variants. Nanopore data could resolve the breakpoints in all samples, and due to the length of nanopore reads, allele phasing was also possible. Specifically, the phasing results allowed for the determination of founder events, which could be used to decipher variant ancestry.
Furthermore, Kieran and colleagues re-sequenced samples which they had previously analysed by whole-genome bisulfite sequencing (WGBS) using a short-read technology and saw a good correlation between these data and Oxford Nanopore methylation calling. Specifically, Kieran’s group performed tumour versus normal differential methylation analysis of brain clinical research samples. In one Lynch syndrome sample, the promoter of MLH1 was clearly differentially methylated, and phasing, using the long nanopore reads, revealed a clear allelic separation of differential methylation.
Kieran further shared that his team is currently looking into allelic transcription. Using the phase blocks obtained from long nanopore reads, gene expression, methylation, and CNV data can be mapped to its corresponding allele, and, potentially, the cause of differential expression can be determined.
‘Nanopore long-read sequencing improves resolution of structural variants, phasing, and oncoviral integrations, plus you get 5mc and 5hmc methylation data with no added cost.’
Validating tumoroid genomes with long nanopore reads
The utility of Oxford Nanopore long reads was further demonstrated at London Calling by Federica Di Maggio, from CEINGE-Advanced Biotechnologies, Naples, Italy. Federica and her group have focused their research on colon cancer, for which they are pioneering a new method for establishing tumour-like organoids, or tumoroids, to reproduce the disease in vitro. The use of tumoroids may allow efficacy studies into new types of therapies in vitro.
Naturally, validation that the tumoroids are mimicking the actual tumours is critical for the potential use of this method in research. To this end, the team performed experiments with DNA extracted from the tumoroids, tumour tissue research samples, and samples of paired healthy tissue. These experiments included a 58-gene short-read colon cancer panel – but what about the tumoroid genome outside of this panel? As Federica put it, ‘obviously, we did not want to focus our attention just on these 58 genes’.
Therefore, Federica and her team performed WGS of 48 samples with their PromethION 24 device. As their PromethION yielded >20x depth of coverage per sample, they now intend to use this data to perform SNV calling, SV calling, and methylation profiling. When presenting at London Calling, Federica and her group had already analysed two samples, which confirmed a good correlation with the mutations found in the tumoroids and tumour tissue.
Overall, the research presented by Abderaouf, Kieran, and Frederica, plus many more scientists at this year’s London Calling conference, highlighted how accessible Oxford Nanopore sequencing is enabling the detection of multiple variant types, advancing their research into cancer.
*London Calling 2022 hybrid conference, hosted by Oxford Nanopore Technologies; May 18–20, 2022
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