With a wealth of genomic technologies rapidly advancing with falling cost, implementing genomics into trials and routine care is becoming much more economically viable. However, choosing the right technology for a research question is not always as simple as asking: “what is the most powerful technology available now?.” With cost and clinical considerations, reporting, computing power, and staff expertise all playing a role in the decision-making process, many trials continue to use one technology over the other.
In this article, we will give a brief overview of the pros and cons of different technologies and an insight into the views of our panellists from our upcoming virtual discussion: Promises and pitfalls of precision medicine clinical trials, now and in the future.
Technology overview:
Whole genome sequencing is a powerful tool that can capture a wealth of information and given the falling cost of sequencing technologies, it is now a feasible option for integration into clinical trials. Advantages of Whole Genome Sequencing include:
- Whole genome sequencing (WGS) allows examination of SNVs, indels, structural variants, and CNVs in coding and non-coding regions of the genome. It omits regulatory regions such as promoters and enhancers.
- WGS has more reliable sequence coverage. Differences in the hybridisation efficiency of WES capture probes can result in regions of the genome with little or no coverage.
- Coverage uniformity with WGS is superior to whole exome sequencing (WES). Regions of the genome with low sequence complexity restrict the ability to design useful WES capture baits, resulting in off-target capture effects.
- PCR amplification is not required during library preparation reducing the potential of GC bias. WES frequently requires PCR amplification as the bulk input amount needed to capture is generally ~1 ug of DNA.
- Sequencing read length isn’t a limitation with WGS. Most target probes for exome-seq are designed to be less than 120 nt long, making it meaningless to sequence using a greater read length.
- A lower average read depth is required to achieve the same breadth of coverage as WES.
- WGS doesn’t suffer from reference bias. WES capture probes tend to preferentially enrich reference alleles at heterozygous sites producing false-negative SNV calls.
- WGS is more universal. If you’re sequencing a species other than human your choices for exome sequencing are pretty limited.
Whole exome sequencing sequences all the protein-coding regions of the genome. Advantages of Whole Exome sequencing include:
- If your study is discovery-based, i.e. you don’t know what genes you need to target, WES is the obvious choice.
- Exome panels are commercially available, they don’t need to be customised or designed.
- Exome sequencing services are fairly standard, and have a standard cost range.
Targeted gene sequencing panels are tools used to analyse specific mutations in a given sample. They contain a select set of genes or gene regions that have known or suspected associations with the disease or phenotype under study.
- Focusing on individual genes or gene regions allows you to sequence at a much higher depth than exome-seq, e.g. 2,000-10,000x as opposed to 200x which is typical with exome-seq.
- High depth sequencing enables the identification of rare variants
- Can be customised for different samples types, e.g. FFPE, cf/ctDNA, degraded samples.
- Lower input amounts can be used with targeted gene panels (1 ng vs. 100 ng with whole exome sequencing).
- Gene panels can be customized to only include genomic regions of interest. Why sequence everything when you don’t need that extra information?
- Gene panel workflows are a lot simpler and time to results is often as little as 1-2 days.
- You can process thousands of samples on a single sequencing run. Targeted gene panels can be run at a higher throughput and are often more cost-effective than whole exome sequencing.
We asked the panellists of our virtual discussion what their thoughts were .We asked: as technology is moving forward, many people think that we can just sequence the entire genome of patients and use this wealth of information for clinical trials. Why would you choose to do panel sequencing?
Their answers were:
Pauline Rehal: The actual technical cost of sequencing is coming down, whether that is for whole genome or small/large targeted panels. NGS services are now well established in many laboratories. Therefore, sequencing is now considered more accessible. However, when people talk about the cost of sequencing, they do not consider the whole cost i.e. from sample receipt to report. The larger the panel the higher the number of variants that will be detected. The burden of work has shifted from the technical aspect of sequencing to the analytical and interpretation end. Sophisticated bioinformatics are needed for analysis and interpretation. Commercial interpretation tools are available but are costly. The other challenge is the sample from the tumour. The 100KGP attempted to use FFPE (Formalin-fixed paraffin-embedded), which is the standard for pathology, but had to move to fresh frozen due to the quality issues and consequent incompatibility with whole genome sequencing. Large panel testing overcomes the FFPE issue and makes variant interpretation more manageable. However, its limitation is the breadth of information, panels must be designed to pick up specific variants. An option is performing WGS but applying virtual analysis panels to limit variant interpretation to the most relevant targets. The advantage is that the raw data is still available to be re-analysed to answer the new questions as they arise.
Alastair Greystoke: When you sequence the whole genome, you will commonly find multiple abnormalities, and that can actually lead to additional problems. One can be working out what the primary driver is in that patient for cancer, and what may be most likely to respond to targeted therapy. You can also find incidental findings that can be unexpected, both in terms of the cancers genomics but also unrelated to the cancer, and this may have implications both for them and their families health.
There are also a number of patients’ tumours that may not be suitable for whole-genome sequencing, however where you may be able to get the answer using a simpler technique. This could be a small next-generation sequencing panel aimed at abnormalities that you can target or using a liquid biopsy on circulating free DNA which may give you a more rapid result and enable you to match the patient up with appropriate targeted treatment.
Maria Cerone: The two approaches have different applications and strengths. From a clinical perspective, panel sequencing can give all the information needed for patient stratification at a very good depth of reading. It is also cheaper and less labour intensive to do and can use FFPE samples from leftover diagnostic biopsies. From a research perspective, WGS gives more comprehensive information that can be used to increase the knowledge and understanding of the disease.
Donald White: Panel sequencing gives us the depth we require when we need confidence in calling genes harbouring no aberrations, as in the case of molecular exclusion for trial arms. As it is less labour intensive, it is a good option for some trials.
Don’t forget to join the discussion on the promises and pitfalls of precision medicine clinical trials now and in the future on Thursday 11th June, 4pm BST.
