When to use targeted resequencing — choosing the right NGS method
Simon Hughes and Dan Swan
Next generation sequencing (NGS) is continuing to revolutionise genetic screening, with ever-evolving techniques becoming relatively inexpensive, fast and widespread. What once took years can now be performed in a matter of days or even hours, enabling investigators to simultaneously screen for tens of 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.
NGS permits the detection of unbalanced chromosomal rearrangements, sub-chromosomal deletions or duplications, loss of heterozygosity, SNPs, and indels, as well as the more difficult to detect copy-neutral variants (e.g. balanced chromosomal inversions or translocations). As costs decrease and informatics improve, NGS is an increasingly powerful and accessible tool, particularly in the identification of underlying genetic aberrations contributing to diseases such as cancer.
With such proliferation of NGS technologies and methods, it may not always be immediately obvious which approach is the most suitable for a given study. While whole genome sequencing (WGS) provides the most comprehensive data, it is not a cost-effective technique for identifying rare variants such as those found in heterogeneous cancer samples. It also generates high volumes of data, which makes analysis particularly complex and resource-intensive.
To avoid the hurdles associated with WGS, more focused targeted resequencing experiments can be implemented, such as whole exome sequencing (WES) and targeted panel sequencing (TPS). Targeted resequencing methods offer powerful and economical alternatives, allowing in-depth investigation of regions of interest.
This whitepaper aims to examine the various targeted resequencing strategies available and explore when they are appropriate, in order to aid investigators in choosing the most suitable NGS method.
|Whole genome sequencing||- Sequencing of the entire genome|
|Targeted resequencing||- Sequencing of selected regions of the genome (i.e. whole exome Sequencing or targeted panel sequencing)|
|Whole exome sequencing (WES)||- Sequencing of all exons within the genome|
|Targeted panel sequencing (TPS)||- Sequencing of typically smaller selected regions of the genome using pre-designed (off-the-shelf) or custom panels|
|Depth of coverage/ read depth||- The number of times DNA is sequenced to ensure data accuracy|
|Uniformity of coverage||- How equal the depth of coverage is across all regions|
|Sensitivity||- Percentage of targets captured|
|Specificity||- Percentage of on target sequences|
Why choose targeted resequencing over whole genome sequencing?
WGS allows detection of all possible variants including CNVs, indels and SNPs without the need for a priori information and is therefore particularly useful for discovery. In contrast, targeted approaches require some prior assumptions of genetic content to ensure that relevant variants are not missed. Although WGS produces the most comprehensive data, there are several cost, time and data analysis disadvantages.
Targeted resequencing offers various benefits over WGS. It enables increased focus on the areas most likely to provide relevant data. Reduced target size raises the depth of coverage which enhances sensitivity, increasing the chance of finding biologically relevant variants. This makes targeted resequencing especially useful for investigating disease. Focusing on specific regions linked to disease can drive therapeutic intervention while also reducing the amount of unsolicited findings or superfluous data.
Such 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 and some cancers.6,7 There has been success in using WES for complex disorders8 and TPS in assessing personal disease risk.9
In addition to enhanced coverage and simpler data analysis, further advantages offered by targeted resequencing are cost and time benefits, making the technique more accessible. The opportunity to increase sample throughput increases costeffectiveness while the short turnaround time can be further enhanced by the use of benchtop sequencers such as the Illumina MiSeq™, which offers a sequencing time of approximately 27 hours in comparison to an Illumina HiSeq™ run of 11 days. Table 1 provides further comparative data on the various NGS methods.
Recent market research surveys have 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).
* Using Illumina HiSeq™ 2000.
Table 1: Comparison of WGS, WES and TPS methods. Depth of coverage and multiplexing capacities increase with more focused targeting, while speed of analysis and corresponding cost decrease.
Figure 1: Market Research Survey conducted by OGT in April 2013 showing the NGS method of interest of 596 respondents.
What to target?
Once targeted resequencing has been selected, there remains a choice of how tightly to set the focus. WES selectively analyses the coding regions (i.e. exons) of the genome, while TPS offers flexibility in choice of regions to be targeted.
The human exome is just 1.5% of the 3 billion base pairs that make up the human genome and includes 85% of all disease-causing mutations.3 This makes WES an excellent choice for investigators of disease to increase depth of coverage and causal variant detection without compromising the chance of discovering de novo mutations. If the biological question is more focused however, TPS offers significant advantages. Custom designs can provide information on both intronic and exonic regions that might be associated with a particular disease, biological pathway or are linked to therapeutic intervention. This opens up the potential for detecting novel point mutations or targeting of known mutation hot-spots.
The use of TPS facilitates further increased depth of coverage, enhancing detection of rare variants or variants present in highly heterogeneous samples. This is of particular importance in cancer research as such variants may be missed or under-represented in WES. Increased read depth and decreased offtarget noise in TPS enables improved sensitivity and specificity (Figure 2).
Figure 2: Advantages conferred by targeting of sequencing. (A) Reduced hybridisation to off-target regions. (B) Increased sensitivity for detecting sequence variants. (C) Increased coverage for high GC-rich regions
As the design of custom targeted panels is intricate, many researchers chose to use service providers to facilitate this process. Design requires a delicate balance between sensitivity, specificity and uniformity of coverage. Low uniformity creates sensitivity issues resulting in under-sequenced regions. Service providers can effectively manage this whole process with dedicated molecular biology and bioinformatics expertise, freeing the investigator to concentrate on interpretation of results
How to Target — Which Capture Technology?
An integral part of targeted resequencing methods is the choice of target-enrichment capture technology used, which is dependent on several factors such as concentration of starting DNA available, size of region of interest and specificity requirements. However, it should be taken into account that the quality of results generated using differing capture technologies can vary widely.
The two major capture technologies are ampliconbased methods and in-solution capture. While amplicon-based methods are much faster than insolution hybridisation, uniformity of coverage is superior for in-solution capture. This is important as low uniformity can have a negative impact on variant detection. In addition, amplicon-based methods can miss variants if they lie within priming sites.
Choosing the right sequencing method essentially comes down to the needs of the individual project. However, for projects focused on identifying causal and rare variants, particularly related to disease, targeted resequencing methods offer logical, costand time-effective choices.
While WGS generates large amounts of data, it is not necessarily better to sequence more of the genome. Indeed the argument exists that many findings from whole genome studies may have been found faster, more cost effectively and with less data complexity using a WES approach. TPS provides an extension of this by offering a further streamlined, logical and focused approach. Both targeted resequencing methods offer insightful, cost-and time-effective analysis for genomic regions of interest without the burden of the tremendous amount of data generated by WGS.
Whole Exome Sequencing
- Good for investigating diseases
- 85% of disease causing mutations are found in exon regions3
- Good coverage of all exons allows detection of causal or de novo variants
- No coverage of intronic regions
- Decreased computational complexity of analysis than WGS (but still challenging)
- Higher sample throughput than WGS and increased multiplexing opportunities
- Increased cost-efficiency compared with WGS
Targeted Panel Sequencing
- Sequencing of a selection of specific genes or a specific region of the DNA
- Allows researchers to focus on genes e.g. which are specific to disease or biological pathway
- Detects rare variants or variants present in highly heterogeneous samples
- Increased read depth and decreased off-target noise provide improved sensitivity and specificity
- Decreased complexity of computational analysis
- Able to optimise design for 100% coverage of target region, including exonic and intronic regions not covered by WES
- Increased focus may cause some variants to be missed
- Higher sample throughput with increased multiplexing opportunities than WES
Capture technology should also be considered when choosing an NGS approach as results can vary between the technologies. In-solution techniques are often preferred due to their superior uniformity of coverage.
The complexity of bait design and the volume of data generated by NGS techniques make the use of NGS service providers with dedicated molecular biology and bioinformatics expertise a somewhat logical choice for researchers. These providers bear the significant expense of keeping up to date with the newest equipment and ensure that staff are appropriately trained. They also implement sophisticated laboratory information management systems (LIMS) and maintain the latest analysis techniques. Outsourcing sequencing requirements can free researchers to focus on interpreting meaningful results, by utilising the expertise of external providers in study design through to data analysis.
Oxford Gene Technology (OGT) is a leading genomic services provider founded by Professor Sir Ed Southern. The OGT Genefficiency™ genomic services offer flexible, comprehensive next generation whole exome, pre-designed panel (e.g., SureSeq Solid Tumor Sequencing Panel) or custom panel sequencing. OGT has significant expertise in probe hybridisation dynamics, and custom panel sequencing services include complimentary bait design, which delivers significantly improved capture efficiency and uniformity of sequencing.
Genefficiency™ covers the whole NGS pipeline, from project conception through to the delivery of high-quality, meaningful results (Figure 4). OGT has developed a user-friendly, unique and complimentary software package that allows users to rapidly filter through to the results that are relevant to the project, overcoming the common bottleneck of handling the huge data sets produced by NGS applications. Through an easy-to-use, mouse-click navigation system, thousands of variations can be filtered within minutes to just a handful requiring further validation — with no requirement for additional bioinformatics resource (Figure 5).
Figure 4: The OGT Genefficiency™ services pipeline. OGT offers complete management of the process from fully optimised project and capture design through to the delivery of an intuitive, user-friendly report, providing investigators with easy access to meaningful data.
Figure 5: Rapid variant filtering using the OGT NGS software. Data from Tariq et al (2011)10 were analysed using the OGT NGS pipeline. The results, filtered by read depth, homozygosity, novelty and predicted consequence on the protein, highlight a causative mutation in this exome sample.
Specifically designed for cancer, the new SureSeq™ Solid Tumour Profiling Sequencing Service is a highly sensitive and specific solid tumour sequencing service that delivers discovery of novel and known somatic variants from fresh, frozen and FFPE tissue samples. The 58-gene panel targets the full coding sequences of cancer-related genes, allowing discovery of all variants present in these genes, not just in known hotspots. The panel content was driven by the Technology Strategy Board’s Stratified Medicines Programme in conjunction with Cancer Research UK, as well as prioritised cancer-related genes from the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Results are delivered back in an interactive report which can be run on a PC with no requirement for additional software or local bioinformatics support.
- 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. [Epub ahead of print]
- 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
- 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
- Ng, S.B. et al (2010) Exome sequencing identifies the cause of a mendelian disorder. Nature Genetics 42, 30-35
- Ng, S.B. et al (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461, 272-276
- Wei, X. et al (2011) Exome sequencing identifies GRIN2A as frequently mutated in melanoma. Nature Genetics 43, 442-446
- 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
- Lehne, B. et al (2011) Exome localisation of complex disease association signals. BMC Genomics 12:92
- Klassen, T. et al (2011) Exome Sequencing of Ion Channel Genes Reveals Complex Profiles Confounding Personal Risk Assessment in Epilepsy. Cell, 145, 1036-1048
- Tariq, M. et al (2011) SHROOM3 is a novel candidate for heterotaxy identified by whole exome sequencing. Genome Biology 12, R91
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