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Nucleic acids research2023; 51(13); e69; doi: 10.1093/nar/gkad460

A method to generate capture baits for targeted sequencing.

Abstract: Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Here, we describe a DNA bait synthesis approach for hybridization capture that we call Circular Nucleic acid Enrichment Reagent, or CNER (pronounced 'snare'). The CNER method uses rolling-circle amplification followed by restriction digestion to discretize microgram quantities of hybridization probes. We demonstrate the utility of the CNER method by generating probes for a panel of 23 771 known sites of single nucleotide polymorphism in the horse genome. Using these probes, we capture and sequence from a panel of ten ancient horse DNA libraries, comparing CNER capture efficiency to a commercially available approach. With about one million read pairs per sample, CNERs captured more targets (90.5% versus 66.5%) at greater mean depth than an alternative commercial approach.
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Publication Date: 2023-06-01 PubMed ID: 37260085PubMed Central: PMC10359599DOI: 10.1093/nar/gkad460Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article presents a new method called Circular Nucleic acid Enrichment Reagent (CNER) for creating DNA bait for hybridization capture, particularly useful in genomic analyses where traditional methods often fail. The study illustrates CNER’s effectiveness through a comparison with a commercial method, demonstrating its superior capture efficiency in sequencing from ancient horse DNA samples.

Introduction to the Circular Nucleic acid Enrichment Reagent (CNER) Method

  • The new DNA bait synthesis technique for hybridization capture is called Circular Nucleic acid Enrichment Reagent (CNER).
  • The article emphasizes the use of CNER in situations where target content is low, DNA is fragmented, and multiplex PCR or other targeted techniques often fail. This makes it especially useful in the analysis of genomic data from ancient, environmental, and forensic samples.
  • The CNER method leverages rolling-circle amplification followed by restriction digestion to break down microgram quantities of hybridization probes.

Application and Testing of the CNER Method

  • The researchers tested CNER’s utility in creating probes for a panel of 23,771 known sites of single nucleotide polymorphism in the horse genome.
  • These probes were then employed to capture and sequence from a panel of ten ancient horse DNA libraries. The capture efficiency of CNER was evaluated in comparison to an existing commercially available approach.

Comparison with Alternate Approaches

  • The effectiveness of CNER was compared to a commercially available method regarding capture efficiency and mean depth.
  • On the performance metrics, the CNER method outperformed the commercial alternative. With approximately one million read pairs per sample, CNER captured more targets, achieving 90.5% success rate as opposed to 66.5% with the commercial method.
  • Furthermore, CNER also managed to capture targets at a greater mean depth than the commercially available method. This superior capture depth implies that CNER might be better at capturing and sequencing from difficult sources such as the horse DNA libraries mentioned in this study.

Cite This Article

APA
Sundararaman B, Vershinina AO, Hershauer S, Kapp JD, Dunn S, Shapiro B, Green RE. (2023). A method to generate capture baits for targeted sequencing. Nucleic Acids Res, 51(13), e69. https://doi.org/10.1093/nar/gkad460

Publication

Nucleic acids research
ISSN: 1362-4962
NlmUniqueID: 0411011
Country: England
Language: English
Volume: 51
Issue: 13
Pages: e69

Researcher Affiliations

Sundararaman, Balaji
  • Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
  • Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
Vershinina, Alisa O
  • Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
Hershauer, Samantha
  • Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
  • Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
Kapp, Joshua D
  • Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
Dunn, Shelby
  • Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
Shapiro, Beth
  • Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
  • Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
  • Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
  • UCSC Genomics Institute, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
Green, Richard E
  • Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
  • UCSC Genomics Institute, University of California Santa Cruz, Santa Cruz, CA 95064, USA.

MeSH Terms

  • Animals
  • Horses / genetics
  • DNA / genetics
  • Genomics
  • Sequence Analysis, DNA / methods
  • Nucleic Acid Hybridization / methods
  • High-Throughput Nucleotide Sequencing / methods

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Citations

This article has been cited 5 times.
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  3. Sun T, Li S, Liu Y, Zhou K, Wang J, Lei Z, Guo X, Xing J, Guo W. Comparative evaluation of DNA and RNA probes for capture-based mitochondrial DNA next-generation sequencing. BMC Biol 2025 Aug 8;23(1):248.
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