Targeted Sequencing: Choosing Between Hybridization Capture and Amplicon Sequencing

Targeted sequencing is an effective way to sequence and analyze specific genomic regions of interest. This method enables researchers to focus their efforts on their desired targets, as opposed to other methods like whole genome sequencing that involve the sequencing of total DNA. Utilizing targeted sequencing is an attractive option for many researchers because it is often faster, more cost-effective, and only generates applicable data. While there are many approaches to targeted sequencing including molecular inversion probes and adaptive sampling, the two most prominent methods, hybridization capture and amplicon sequencing, will be covered in this article.

Advantages of targeted sequencing
One of the main advantages of targeted sequencing is that it reduces costs. This is because targeted sequencing experiments only generate sequencing reads for the regions of interest, which lowers the data requirements per sample and increases the ability to scale and highly multiplex the sequencing run. The focus on specific targets and reduction of reads per sample also allows researchers to deep sequence or “resequence” important areas within the genome. Deep sequencing is important for research investigating low-frequency mutations or rare cell types.

In addition, the analysis process of targeted sequencing data is much faster and easier than traditional sequencing methods. The reads generated from successful targeted experiments can be quickly aligned to references of the known target regions. Targeted sequencing experiments also have higher flexibility and can be customized to perform a wide range of applications. With many available commercial offerings, users can design their own targeted sequencing product from existing methods to meet the needs of their study.

Limitations of targeted sequencing
Despite the utility of targeted sequencing, it also has several limitations. Primarily, regions not included in the design of the targeted method will not be enriched and sequenced. While the aim of targeted experiments is to only sequence the regions of interest, it also limits researchers from investigating anything outside the direct scope of their study. Furthermore, targeted sequencing requires knowledge of the regions of interest; otherwise, primers and probes cannot be designed.

Lastly, mutations in or around the target regions can potentially cause problems during hybridization or amplification. Primers and probes that fail to bind lead to uneven coverage or a failure to sequence important targets. This is particularly a concern in targeted applications that are used to detect variants or infectious diseases.

Hybridization capture
Hybridization capture is a targeted sequencing method that uses oligonucleotide probes (also referred to as baits) to capture regions of interest through the hybridization of complementary sequences. The process begins by first enzymatically or mechanically fragmenting the input DNA and then capturing the regions of interest using complementary probes. Common capture methods use biotinylated DNA probes that isolate, or “pull down”, the target sequences using streptavidin magnetic beads. Adapter sequences are then added to the isolated sequencers and they are optionally amplified. The sequencing libraries are then pooled and loaded onto the appropriate sequencer.

Each of the steps included in the hybridization workflow may occur differently depending on the panel’s design and the sequencing instrument being used. Hybridization capture remains a popular method for targeted sequencing due to its robustness and high uniformity coverage of target regions. Researchers have successfully used this method for viral surveillance¹, identifying deleterious mutations², and guiding clinical treatment decisions for cancer patients³.

Common applications
• Exome sequencing
• Cancer research
• Liquid biopsy and minimal residual disease
• Rare variant detection
• Infectious disease

Amplicon sequencing
Amplicon-based targeted sequencing utilizes PCR with primers flanking the regions of interest to amplify specific targets. This is often done through multiplex PCR, and the resulting products (amplicons) are converted into sequencing libraries through the addition of adapters. The final libraries are then pooled and loaded on the appropriate sequencer.

Amplicon sequencing has remained an essential targeted sequencing method due to its ease of use and flexibility. This method has been used to increase our understanding of the microbiome⁴, detect infectious disease⁵, and monitor metastatic breast cancer⁶.

Common applications
• Metagenomics (16s rRNA and ITS1)
• CRISPR screening
• Genes associated with inherited disorders
• SNPs and small indels
• Molecular breeding and genotyping

Choosing a targeted method

Hybridization capture
• Hybridization capture is often best for larger targets and applications that require investigating longer genomic regions.
• Workflows for hybridization capture typically allow for a higher number of targets, while the multiplex PCR required for amplicon sequencing has more limitations on the number of amplicons it can produce.
• PCR is not always required for hybridization capture experiments, so workflows without amplification won’t introduce PCR errors. This is a common concern when performing variant analysis.
• Amplification bias and dropouts during amplicon sequencing can cause an uneven distribution of amplicons and impact coverage.

Amplicon sequencing
• Amplicon sequencing is best for smaller targets and is often the method of choice when dealing with more difficult samples, like low-input or degraded DNA.
• This method is typically much faster and has fewer steps than hybridization capture, which may require overnight incubation steps.
• Amplicon sequencing is often cheaper than hybridization capture due to the simple reagent requirements for primers and PCR mix.
• Compared to hybridization capture, amplicon sequencing tends to have a higher on-target rate.

Recommendations for both methods
Regardless of which strategy is used for the targeted sequencing experiment, the chosen method should include appropriate controls. Without proper controls, it would be impossible to discern whether there was an absence of a particular target or an experimental failure. Sequencing controls are one of the important qualities of targeted products, according to Patrick Finn, President and Chief Operating Officer at Twist Bioscience.

“You want to have exquisite controls and good solid references against any complicated assay you're doing in any sequencing experiment,” said Finn. “Twist offers a robust, efficient target enrichment workflow to allow you to enrich whatever is of interest to you – from small to large panels, customized to the project. And, we offer a suite of controls to validate that your assay is working properly.”

The use of controls is also particularly important when using targeted sequencing to confirm the presence of a particular mutation or pathogen. Controls used in amplicon sequencing experiments show proper amplification of targets, while controls in hybridization capture experiments ensure proper capture efficiency. The control sequences used should be a sequence present in each of the samples or, in some cases, a spike-in control.

Improvements to hybridization capture and amplicon sequencing over the years have increased their capabilities and the breadth of their applications. Many of the previous constraints of these methods are no longer applicable, and newer products have been developed to handle a wider range of applications. One such product was highlighted by Dr. Bjoern Textor, Senior Application Manager at NEB. “The NEBNext Direct Genotyping Solution combines the benefits of capture-based enrichment and amplicon sequencing. Capture-based assays are easier to scale in content, while amplicon-based capture has a faster turnaround time. NEBNext Direct combines these benefits in a capture-based assay.”

Both targeted methods are compatible with all of the available sequencing technologies (i.e., short- and long-read instruments), but protocols may need to be tailored to produce libraries suitable for the appropriate sequencer. In many cases, the solution for your targeted experiment may already exist—there are many commercial options available for a variety of popular applications.



References

1. Wylie TN, Wylie KM, Herter BN, Storch GA. Enhanced virome sequencing using targeted sequence capture. Genome Research. 2015;25(12):1910-1920. doi:https://doi.org/10.1101/gr.191049.115
2. Nectoux J, de Cid R, Baulande S, et al. Detection of TRIM32 deletions in LGMD patients analyzed by a combined strategy of CGH array and massively parallel sequencing. European Journal of Human Genetics. 2014;23(7):929-934. doi:https://doi.org/10.1038/ejhg.2014.223
3. Rozenblum AB, Ilouze M, Dudnik E, et al. Clinical Impact of Hybrid Capture-Based Next-Generation Sequencing on Changes in Treatment Decisions in Lung Cancer. Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer. 2017;12(2):258-268. doi:https://doi.org/10.1016/j.jtho.2016.10.021
4. Gupta S, Mortensen MS, Schjørring S, et al. Amplicon sequencing provides more accurate microbiome information in healthy children compared to culturing. Communications Biology. 2019;2(1). doi:https://doi.org/10.1038/s42003-019-0540-1
5. LaVerriere E, Schwabl P, Carrasquilla M, et al. Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: A malaria case study. Molecular Ecology Resources. 2022;22(6):2285-2303. doi:https://doi.org/10.1111/1755-0998.13622
6. Gao M, Callari M, Beddowes E, et al. Next Generation-Targeted Amplicon Sequencing (NG-TAS): an optimised protocol and computational pipeline for cost-effective profiling of circulating tumour DNA. Genome Medicine. 2019;11(1). doi:https://doi.org/10.1186/s13073-018-0611-9