Target Enrichment Strategies for Genomic Research

“Target enrichment for next-generation sequencing (NGS) refers to the process of capturing specific regions of interest in a genome or transcriptome for focused sequencing,” explained Mark Andersen, Senior Director of R&D at Thermo Fisher Scientific. “This approach is commonly used to study genetic variations in specific regions such as biomarkers in disease-associated genes.”

Several target enrichment methods have been developed over the years, with the primary two being hybridization-based capture and amplicon-based PCR. Hybridization capture workflows use oligonucleotide probes to capture regions of interest through the hybridization of complementary sequences, while amplicon-based enrichment strategies employ PCR with primers flanking the target regions to amplify them. “By focusing sequencing on specific regions, target enrichment can improve efficiency, reduce cost, increase the depth of coverage in the regions of interest, and simplify analysis and reporting,” Andersen noted.

Pedro Echave, Senior Manager at Revvity, also highlighted the benefits of target enrichment with an apt example. “Most human genetic disorders are caused by changes in exons, which constitute 1.5% of the human genome. Target enrichment allows sequencing of the exons of interest instead of sequencing the whole genome of an individual, so the economic advantage is obvious.” Echave further explained that target enrichment enables users to sequence at much higher depths (1,000x or higher), which allows the identification of rare variants. This is particularly important in applications like cancer research that require deep sequencing to identify low levels of somatic mutations, which can only be achieved using target enrichment.

Choosing a target enrichment method

The significant increase in available enrichment options in recent years has provided more opportunities for researchers but has made selecting the right approach more challenging. “There are some caveats and best practices customers should be aware of when choosing between the different target enrichment modalities,” explained Ellie Juarez, Ph.D., NGS Business Unit Manager at Integrated DNA Technologies. “Factors include the number of targets being interrogated, the hands-on time of the full workflow desired, the need for bioinformatics analysis and tools, and the type of variants or anomalies the scientist plans to interrogate.”

Jon Sherlock, Senior Director of Product Management at Thermo Fisher Scientific, also shared that when choosing a target enrichment solution, “it is [just] as important to consider the panel design as it is to consider the other details related to the assay to ensure the relevant regions are targeted adequately, maximizing the sensitivity and specificity of the test, while avoiding off-target enrichment and false positive results.” Once the optimal design is established, then it’s important to evaluate the downstream NGS process, particularly the sample type and input, to ensure that the protocol and platforms meet the requirements of the end user.

In addition to these considerations, Echave provided a comparison of the two primary enrichment methods. Hybridization capture allows the profiling of different variant types and the selection of many genes, including full exomes, but it is typically more complex. In contrast, amplicon sequencing is quick, straightforward, and ideal for analyzing a small number of genes and focusing on single nucleotide variants and indels. However, Echave pointed out that the success of both methods is dependent on the quality of the primer or probe design, as poor design can lead to significant gaps in the data and sequencing of off-target regions.

To further refine the selection of an appropriate enrichment method, Giorgio Pea, Product Manager at Thermo Fisher Scientific, suggested considering several critical factors. First, assess the compatibility of the enrichment method with the starting material you plan to use. Next, evaluate the flexibility and ease of panel design, as well as determine if the technology can be used for other applications. Additionally, review the turnaround times and whether your workflow requires automation for this process. The quality control measures, special analyses, and reporting requirements of your lab should also play an important role in your decision-making process. Lastly, consider the level of services and support available for the enrichment product within your institution. This will ensure a streamlined and successful implementation of the technology.

Recent advancements and innovations

“With the advent of new sequencing technologies and companies, new and interesting approaches to target enrichment continue to emerge,” stated Juarez. Among these advancements, Juarez pointed to an innovative on-sequencer enrichment method by Element Biosciences, as part of the IDT Collab Network, which simplifies targeted sequencing efforts. The partnership between the two groups led to the development of new adapters, universal blockers, and library amplification primer mixes designed for native library construction and hybridization capture enrichment on the AVITI™ system. Furthermore, IDT is offering tailored design solutions and unique long-read capture methods that integrate with long-read sequencing technologies from companies like Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio).

Recent innovations in auxiliary products have also significantly enhanced the target enrichment process. Andersen shared that some of the developments from his group include enhancements in automation, reverse transcription, sample ID, and library normalization. “These innovations have enabled our customers to expand their studies into new directions, such as performing NGS tests with ultra-low limits of detection,” he emphasized. In addition, Sherlock expressed enthusiasm about the increased adoption of targeted NGS for diverse uses and applications like DNA methylation analysis and pharmacogenomics research. He is optimistic about the increasing adoption of targeted enrichment-based NGS assays in clinical settings.

One of the notable advancements in target enrichment is the identification of structural variants (SVs), Echave explained. This includes changes in regions of the genome such as deletions, duplications, and repetitive sequence expansions over 50 base pairs. Traditionally, short-read sequencing technologies have struggled to detect these regions; however, recent developments in long-read sequencing platforms have greatly improved the ability to identify all types of SVs with high sensitivity and reduced false positives. “More importantly, these techniques allow the identification of SVs within repetitive elements and segmental duplications that were not available with short reads,” Echave noted. These innovative platforms have been commercialized by companies like ONT and PacBio, and despite some current limitations, the potential of these technologies makes them an exciting area for future developments.

Another promising area is the application of CRISPR for target enrichment. “CRISPR-based genome editing can cleave in specific DNA sequences, which should facilitate resolution of homologous genes, detection of various types mutations (even at low frequency) and SV,” stated Echave. This method enables the isolation of target regions without the use of amplification and allows the analysis of genetic and epigenetic profiles for important clinical applications.¹

Each of these developments in target enrichment highlights its critical role in advancing genomics research and clinical diagnostics. Through reduced costs, enhanced efficiencies, and deeper insights into the genome, target enrichment methods for NGS are set to continue transforming the field.

Reference

1. Malekshoar, M., Azimi, S. A., Kaki, A., Mousazadeh, L., Motaei, J., & Vatankhah, M. (2023). CRISPR-Cas9 Targeted Enrichment and Next-Generation Sequencing for Mutation Detection. The Journal of Molecular Diagnostics: JMD, 25(5), 249–262. https://doi.org/10.1016/j.jmoldx.2023.01.010