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Targeted next-generation sequencing techniques: From primer design to sophisticated library construction.

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Τίτλος: Targeted next-generation sequencing techniques: From primer design to sophisticated library construction.
Συγγραφείς: Koryukov M; Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia; Novosibirsk State University, Novosibirsk 630090, Russia. Electronic address: mkoryukov@gmail.com., Mikheeva R; Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia; Novosibirsk State University, Novosibirsk 630090, Russia., Kechin A; Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia; Novosibirsk State University, Novosibirsk 630090, Russia.
Πηγή: Methods (San Diego, Calif.) [Methods] 2026 Sep; Vol. 253, pp. 31-48. Date of Electronic Publication: 2026 May 08.
Τύπος έκδοσης: Journal Article; Review
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Academic Press Country of Publication: United States NLM ID: 9426302 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1095-9130 (Electronic) Linking ISSN: 10462023 NLM ISO Abbreviation: Methods Subsets: MEDLINE
Imprint Name(s): Publication: Duluth, MN : Academic Press
Original Publication: San Diego : Academic Press, c1990-
Ιατρικοί όροι (MeSH): High-Throughput Nucleotide Sequencing*/methods , DNA Primers*/genetics , DNA Primers*/chemistry , Gene Library*, Polymerase Chain Reaction/methods ; Sequence Analysis, DNA/methods ; Humans
Περίληψη: Targeted Next-Generation Sequencing (tNGS) represents a cornerstone technology in modern clinical diagnostics and biological research. Widely adopted by clinical laboratories, tNGS provides a robust and cost-effective alternative to whole-genome sequencing. Despite its extensive applications, the most technically complex and variable stage of the tNGS workflow remains target enrichment, a critical step that ensures target molecules are present at sufficiently high concentrations relative to background sequences. Enrichment methodologies are broadly classified into PCR-based and hybridization-based strategies, each offering distinct advantages and limitations. This review critically examines both approaches within the context of the computational challenges inherent to tNGS assay design, and surveys the full spectrum of established and emerging enrichment techniques, highlighting their suitability across different areas of molecular research and clinical diagnostics. The use of non‑canonical nitrogenous bases, circularization of linear DNA fragments, unique molecular identifiers (UMIs), and partially double‑stranded primers are among the many sophisticated approaches described in the review. We hope this review will contribute to accelerating the development of diagnostic strategies with targeted next-generation sequencing in the rapidly evolving field of molecular diagnostics.
(Copyright © 2026 Elsevier Inc. All rights reserved.)
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contributed Indexing: Keywords: Molecular diagnostics; Mutation detection; Next-generation sequencing; Primer design; tNGS
Substance Nomenclature: 0 (DNA Primers)
Entry Date(s): Date Created: 20260510 Date Completed: 20260613 Latest Revision: 20260613
Update Code: 20260615
DOI: 10.1016/j.ymeth.2026.05.004
PMID: 42107825
Βάση Δεδομένων: MEDLINE
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  Data: Targeted Next-Generation Sequencing (tNGS) represents a cornerstone technology in modern clinical diagnostics and biological research. Widely adopted by clinical laboratories, tNGS provides a robust and cost-effective alternative to whole-genome sequencing. Despite its extensive applications, the most technically complex and variable stage of the tNGS workflow remains target enrichment, a critical step that ensures target molecules are present at sufficiently high concentrations relative to background sequences. Enrichment methodologies are broadly classified into PCR-based and hybridization-based strategies, each offering distinct advantages and limitations. This review critically examines both approaches within the context of the computational challenges inherent to tNGS assay design, and surveys the full spectrum of established and emerging enrichment techniques, highlighting their suitability across different areas of molecular research and clinical diagnostics. The use of non‑canonical nitrogenous bases, circularization of linear DNA fragments, unique molecular identifiers (UMIs), and partially double‑stranded primers are among the many sophisticated approaches described in the review. We hope this review will contribute to accelerating the development of diagnostic strategies with targeted next-generation sequencing in the rapidly evolving field of molecular diagnostics.<br /> (Copyright © 2026 Elsevier Inc. All rights reserved.)
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