The Power of Hybridization Capture: A Guide to Targeted Next-Generation Sequencing

Next-generation sequencing (NGS) has revolutionized genetic research by enabling scientists to sequence millions of DNA or RNA molecules simultaneously. However, the sheer complexity of genetic material often makes it prohibitively expensive to sequence everything with sufficient depth, while also creating bioinformatic challenges during analysis.

Enter targeted sequencing – a strategic approach that focuses on sequencing specific regions of interest in the genome, rather than the entire genome, allowing researchers to achieve deeper read coverage while lowering costs. Among the available targeted sequencing technologies, hybridization capture stands out as a particularly powerful and versatile method.1

Understanding hybridization capture technology

Hybridization capture is a targeted sequencing technique that utilizes customized pools of biotinylated oligonucleotides (known as “baits” or “probes”) to enrich complementary molecules from an NGS library. At the forefront of this technology, is the myBaits® system.

Developed by Arbor Biosciences, myBaits integrates seamlessly into standard NGS workflows.  Here’s how it works:

  1. After library preparation is completed, the pool of double-stranded library molecules is denatured
  2. These molecules hybridize to adapter-specific blocking oligonucleotides
  3. Target-specific biotinylated oligonucleotides, or baits, hybridize to their complementary sequences among the target library molecules
  4. The baits are bound to streptavidin-coated magnetic beads
  5. Off-target library molecules are washed away
  6. Target molecules are released and amplified with universal primers, creating an enriched library ready for sequencing

Key advantages of myBaits hybridization capture technology

Leveraging myBaits hybridization capture delivers a significant reduction in sequencing cost per sample without compromising data accuracy. Further, myBaits hybridization capture provides:

  1. Precision and sensitivity: Exceptional on-target specificity while maintaining target molecule complexity
  2. Novel variant discovery: Detect a full spectrum of mutation types, including SNPs, indels, rearrangements, CNVs, and more
  3. Versatile molecule capture: myBaits probes function independently from the enriched library, enabling them to isolate molecules regardless of their length
  4. Capture elusive targets: Enhanced sensitivity maintains high specificity when isolating rare molecules
  5. Customization and flexibility: Customizable bait design and flexible workflows compatible with any upstream library preparation method and downstream NGS platform

Applications across diverse fields

Researchers across multiple fields are harnessing hybridization capture technology to advance their research with precision and efficiency. Microbiologists and virologists selectively enrich pathogen genomes that might otherwise be overwhelmed by host DNA in clinical samples, transforming previously difficult analyses into routine procedures. Agrigenomic scientists conduct comprehensive genetic studies across diverse crop and livestock species, identifying valuable markers that enhance breeding programs. Environmental DNA researchers leverage the approach to detect target organisms present in extremely low concentrations within complex environmental samples. Evolutionary biologists gain unprecedented insights into genetic relationships, historical population dynamics, and adaptive mechanisms through targeted sequencing. And forensic scientists and ancient DNA researchers use hybridization capture to overcome the inherent challenges of working with compromised and degraded samples.

The applications continue to expand as researchers discover the advantages of focused genomic investigation. To support these efforts, many researchers are turning to customizable solutions, like myBaits, with the versatility to support diverse applications and challenging sample types.

Real world impact of myBaits: research success in action

Researchers at Texas Tech University utilized a universal myBaits panel designed to enrich hundreds of genes from angiosperm species in herbarium specimens. The study demonstrated that even with degraded DNA from herbarium specimens collected decades ago, the panel successfully recovered nearly 200 kb of coding region sequence per species, along with substantial non-coding DNA physically adjacent to the targeted regions. They were able to retain sufficient SNP levels to calculate downstream genetic statistics despite having only 4 samples per species.2

A 2023 study published in Molecular Ecology showcased the power of myBaits in conservation genetics. Researchers studying the critically endangered nightingale reed warblers in the Mariana Islands extracted DNA from museum specimens of extinct species as well as feathers and blood samples from the sole surviving species. Using both nuclear and mitochondrial myBaits panels, they successfully performed detailed analyses of genetic diversity across both extinct and extant species, providing crucial information for conservation efforts of the remaining population.3

These studies and more highlight how researchers are leveraging myBaits technology to overcome complex sequencing challenges and unlock discoveries.

Conclusion: Empowering genomic discoveries with targeted approaches

Hybridization capture represents a powerful approach to targeted next-generation sequencing across agrigenomics, evolutionary biology, microbial sequencing, and other genomic applications. By focusing sequencing efforts on specific regions of interest, researchers are achieving deeper coverage at lower costs, empowering unprecedented discovery opportunity.

For more information about myBaits technology or to explore how myBaits Custom Hybridization Capture Kits can enhance your research.

References:

  1. Beaudry, M. et al. “Enriching the future of public health microbiology with hybridization bait capture” Clinical Microbiology Reviews. (2024) Nov; 37(4). doi: 10.1128/cmr.00068-22
  2. Slimp, M. et al “On the potential of Angiosperms353 for population genomic studies” Applications in Plant Sciences. (2021) May; 9(7). doi: 1002/aps3.11419
  3. Kearns, A. et al. “Conservation genomics and systematics of a near extinct island radiation” Molecular Ecology. (2022) Apr; 31(7). doi: 10.1111/mec.16382

 

Methylation Sequencing: Unraveling the Epigenetic Code with Targeted Solutions

Understanding DNA Methylation: The Epigenetic Cornerstone

DNA methylation is a key epigenetic mechanism controlling gene expression and plays a fundamental role in cellular development, differentiation, and disease progression. The addition of methyl groups to DNA molecules typically occurs at cytosine bases within CpG dinucleotides, altering gene activity without changing the underlying DNA sequence. This epigenetic mechanism serves as an important regulatory system, influencing which genes are expressed or silenced in specific cells.

Aberrant methylation patterns have been implicated in various diseases, particularly cancer, where hypermethylation of tumor suppressor genes or hypomethylation of oncogenes can contribute to disease progression. Advances in developing targeted drugs that modulate DNA methylation have opened promising avenues for epigenetic therapies in oncology. As researchers continue to explore the complex relationship between methylation patterns and human health, methylation sequencing has become critical for understanding these epigenetic landscapes.1

 

The power of targeted methylation sequencing

Comprehensive genome-wide methylation analysis through whole genome bisulfite sequencing (WGBS) represents the premier approach for uncovering methylation patterns across diverse biological samples. While this technique provides a wide breadth of coverage, when research transitions from discovery to targeted validation, the practical utility of WGBS diminishes. High sequencing costs and excessive data generation make WGBS impractical for large sample cohorts and routine clinical analysis where focused investigation is more relevant.

For these studies, targeted methylation sequencing that focuses only on genomic regions of interest emerges as a transformative approach. This method employs the use of probes to bind to specific sequences within a next-generation sequencing library, with subsequent hybridization capture to enrich the library prior to methylation sequencing.

A targeted methylation sequencing approach offers several distinct advantages:

  1. Cost-effectiveness: By focusing on specific regions of interest rather than the entire genome, targeted methylation sequencing significantly reduces sequencing costs while still capturing the most relevant information
  2. Increased sequencing depth: The focused approach allows for higher coverage of target regions, enabling more accurate detection of subtle methylation changes and rare methylation events
  3. Enhanced sensitivity: Targeted methods can detect low-frequency methylation signatures in complex samples, making them ideal for applications like liquid biopsy where informative methylated DNA molecules may be present in low abundance
  4. Streamlined analysis: By limiting analysis to specific genomic regions, targeted approaches simplify data processing and interpretation, accelerating the path from sequencing to meaningful insights

 

myBaits® technology: advancing targeted methylation sequencing

Targeting methylated regions presents unique technical challenges due to the reduced sequence complexity following bisulfite or enzymatic conversion. The myBaits Custom Methyl-Seq system addresses these challenges through a proprietary methylation-specific probe design algorithm that simulates various potential methylation configurations on both strands from converted genomic templates, while thoroughly filtering for specificity. This approach ensures high on-target efficiency while minimizing bias toward either methylated or unmethylated DNA.

The system’s performance metrics are impressive, with custom panels demonstrated to achieve over 80% of reads on-target following hybridization capture, representing an 8000- to 9000-fold enrichment. Furthermore, the system demonstrates compatibility with low-input samples, as little as 1 ng of starting DNA, to maximize sensitivity.

 

Applications in cancer research and beyond

Targeted methylation sequencing has proven particularly valuable in cancer research applications, where methylation signatures can serve as biomarkers for early detection, prognosis, and treatment response.2 Underscoring the clinical potential of targeted methylation sequencing is the rise in cell-free DNA (cfDNA) signatures as a promising tool in oncology. Tumor cells release DNA fragments into the bloodstream that carry distinct methylation signatures, enabling identification and monitoring of tumors through minimally invasive cfDNA-based liquid biopsies rather than traditional tissue biopsies.3 Powerful data presented in a recent webinar, demonstrated the characterization of cancer-associated methylation states in cfDNA. This study leveraged targeted methylation sequencing using the myBaits Custom Methyl-Seq system and revealed accurate measurement of site-specific methylation levels.

Beyond oncology, targeted methylation sequencing supports diverse research areas including developmental biology studies examining dynamic methylation changes during cellular differentiation, neurological research investigating epigenetic contributions to brain development and neurodegeneration, and environmental health studies assessing how environmental exposures influence the epigenome.

 

The path forward with precision epigenetics

As our understanding of epigenetics continues to evolve, targeted methylation sequencing stands as an essential tool for unraveling the complex role of DNA methylation in health and disease. The precision of a hybridization capture approach to methylation sequencing is enabling researchers to interrogate methylation patterns with unprecedented detail, accuracy, and efficiency.

The continued advancement of technologies like Arbor Biosciences’ myBaits Custom Methyl-Seq system promises to further enhance our ability to detect and interpret methylation signatures, ultimately translating epigenetic insights into improved diagnostic approaches and therapeutic strategies.

 

References:

  1. Saghafinia, S. et al. “Pan-cancer landscape of aberrant DNA methylation across human tumors” Cell Reports. (2022) Oct; 39(2). doi: 10.1016/j.celrep.2018.09.082
  2. Liang, G. et al. “DNA methylation aberrancies as a guide for surveillance and treatment of human cancers” Epigenetics. (2017) Mar; 12(6):416–432. doi: 1080/15592294.2017.1311434
  3. Buckley, D. et al. “Targeted DNA methylation from cell-free DNA using hybridization probe capture” NAR Genomics and Bioinformatics. (2022) Dec; 4(4). doi:10.1093/nargab/lqac099
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