NGS– how less can be more
11 Feb 2014 by Evoluted New Media
With the rise of targeted resequencing technologies cutting down on the data analysis bottleneck in next generation sequencing, Dr Simon Hughes discusses what this means in terms of improved accessibility for any sized laboratory
Next generation sequencing (NGS) is a powerful tool for genetic analysis across a wide range of biological research areas, from population studies to identifying disease-linked variants. NGS technologies continue to evolve at an ever-increasing pace, and what once took years can now be performed in a matter of days or even hours, enabling investigators to simultaneously screen for thousands of biologically relevant variants in a single individual. Sequencing of complete genes, chromosomes or even the entire genome can detect known or novel sequence variants at single nucleotide resolution and may provide potential biomarkers of disease. As costs decrease and informatics improve, NGS is becoming an increasingly accessible tool. However, we are still witnessing a transition phase, where for those smaller labs looking to benefit from the latest innovations in NGS, it can be tough to know the most efficient and cost-effective route to harnessing this technology. At the root of the notorious bottleneck of bioinformatics analysis is the tremendous volume of data generated by whole genome sequencing (WGS), the analysis of which is a particularly complex and resource-intensive process. However, for many studies this amount of data is unnecessary. Focusing on targeted resequencing approaches can help to overcome this data analysis hurdle, vastly improving the accessibility of NGS. The term targeted resequencing encompasses two types of approaches, each offering distinct advantages for laboratories wanting to benefit from the advanced and informative technology of NGS. Where whole exome sequencing (WES), as the name suggests, targets the gene-encoding regions of the exome, targeted panel sequencing (TPS) can target any user-defined region of the genome. By reducing the target size and focusing on the regions most likely to yield relevant data, targeted resequencing enables increased depth of coverage, which in turn enhances sensitivity, improving the chance of finding biologically relevant variants. This makes targeted resequencing an efficient approach for investigating disease, as it can reduce the amount of false positives or unnecessary data. In fact, targeted approaches have already had a major impact on disease detection by permitting successful identification of causal mutations for a number of genetic disorders1-5, including cancer6,7. Efficiency translates to savings of both cost and time, making this approach a lot more accessible for a greater number of laboratories, even those not specialising in genomics. Recent market research surveys have even shown that the majority of NGS investigators would choose to use targeted resequencing techniques where possible due to the number of distinct advantages that they offer (Figure 1). [caption id="attachment_36942" align="alignright" width="200"]
Figure 1: Market research survey conducted by OGT in April 2013 showing the NGS method of interest of 596 respondents.[/caption]
The appropriate decision as to which targeted resequencing approach to apply will depend on the individual case. As the exome accounts for only 1.5% of the human genome, and yet includes 85% of all disease-causing mutations3, it’s easy to see how WES methods are highly popular for detecting causal variants without compromising the chance of discovering de novo mutations (particularly where the biological pathway is not fully understood).
In cases where the biological question is more focused, TPS can provide information on specific genomic regions associated with a particular disease that can help drive therapeutic intervention, detect novel point mutations or target known mutation hot-spots. The greater refinement offered by TPS has the potential to enhance detection of rare variants or variants present in highly heterogeneous samples. With increased read depth and decreased off-target noise leading to improved sensitivity and specificity, TPS is ideal in cancer research, where such variants may otherwise be missed or under-represented.
Although targeted resequencing offers a less complex alternative to WGS in terms of data management, a level of bioinformatics expertise is still necessary to get the most out of any one study. Furthermore, the initial experimental design of custom targeted panels is still an intricate process, and for these reasons many researchers take the more straightforward approach of working with a dedicated service provider.
By taking advantage of molecular biology and bioinformatics expertise of companies the whole NGS process from study design through to data analysis is simplified, enabling researchers to concentrate on interpreting results. Indeed, expertise in designing targeted NGS panels can further increase the power of those results by optimising target capture, uniformity of coverage and sensitivity in the NGS process.
In summary, targeted resequencing offers the most logical and efficient NGS option for projects focused on identifying causal and rare variants, particularly related to disease.Although WGS can generate a comprehensive data set, sifting through a mountain of data to identify results relevant to your biological question may not be an effective use of time or resources. In fact, it’s highly likely that many findings from WGS studies would have been found faster and more efficiently using a WES approach and TPS can offer even further flexibility in which specific region to target. The complexity of experimental design and the volume of data generated by any NGS technique makes the use of service providers with dedicated infrastructure a logical choice for researchers. Improving the accessibility of NGS in this way is sure to drive many exciting new discoveries, as investigators are able to harness the power of the latest innovations in NGS, without being an expert in the field.What once took years can now be performed in a matter of days or even hours
References
1. Classen, CF. et al (2013) Dissecting the genotype in syndromic intellectual disability using whole exome sequencing in addition to genome-wide copy number analysis. Human Genetics, April 4. 2. Semler, O. et al (2012) A Mutation in the 5’-UTR of IFITM5 Creates an In-Frame Start Codon and Causes Autosomal-Dominant Osteogenesis Imperfacta Type V with Hyperlastic Callus. The American Journal of Human Genetics 91, 349-357 3. Choi, M. et al (2009) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proceedings of the National Academy of Sciences of the United States of America 106, 19096-19101 4. Ng, S.B. et al (2010) Exome sequencing identifies the cause of a mendelian disorder. Nature Genetics 42, 30-35 5. Ng, S.B. et al (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461, 272-276 6. Wei, X. et al (2011) Exome sequencing identifies GRIN2A as frequently mutated in melanoma. Nature Genetics 43, 442-446 7. Yan, X.J. et al (2011) Exome Sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia. Nature Genetics 43, 309-315 8. Klassen, T. et al (2011) Exome Sequencing of Ion Channel Genes Reveals Complex Profiles Confounding Personal Risk Assessment in Epilepsy. Cell, 145, 1036-1048
Author Dr Simon Hughes, Team Leader Cancer R&D, Oxford Gene Technology (OGT). OGT provides innovative products and services for genome analysis, ensuring the delivery of high-quality, meaningful results.
