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Comparing the Performance of Double-Stranded and Single-Stranded DNA Libraries for Ancient Oral Microbiome Reconstruction.

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Τίτλος: Comparing the Performance of Double-Stranded and Single-Stranded DNA Libraries for Ancient Oral Microbiome Reconstruction.
Συγγραφείς: Burge K; Department of Anthropology, Harvard University, Cambridge, Massachusetts, USA., Velsko IM; Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.; Associated Research Group of Archaeogenetics, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany., Salazar-García DC; Departament de Prehistòria, Arqueologia i Història Antiga, Universitat de València, València, Spain.; Department of Geological Sciences, University of Cape Town, Cape Town, South Africa., Uriarte MH; Departamento de Prehistoria, Arqueología, Historia Antigua, Historia Medieval y Ciencias y Técnicas Historiográficas, Universidad de Murcia, Murcia, Spain., Lomba Maurandi J; Departamento de Prehistoria, Arqueología, Historia Antigua, Historia Medieval y Ciencias y Técnicas Historiográficas, Universidad de Murcia, Murcia, Spain., Warinner C; Department of Anthropology, Harvard University, Cambridge, Massachusetts, USA.; Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.; Associated Research Group of Archaeogenetics, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany.; Faculty of Biological Sciences, Friedrich Schiller University, Jena, Germany.; Department of Human Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA.
Πηγή: Molecular ecology resources [Mol Ecol Resour] 2026 Oct; Vol. 26 (7), pp. e70201.
Τύπος έκδοσης: Journal Article; Comparative Study; Evaluation Study
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Blackwell Country of Publication: England NLM ID: 101465604 Publication Model: Print Cited Medium: Internet ISSN: 1755-0998 (Electronic) Linking ISSN: 1755098X NLM ISO Abbreviation: Mol Ecol Resour Subsets: MEDLINE
Imprint Name(s): Original Publication: Oxford, England : Blackwell
Ιατρικοί όροι (MeSH): Mouth*/microbiology , Metagenomics*/methods , Microbiota* , Gene Library* , DNA, Ancient*, DNA, Single-Stranded/genetics ; Animals ; Humans ; Sequence Analysis, DNA ; Neanderthals
Περίληψη: DNA library construction methods can affect the recovery of ancient DNA, thus influencing downstream analyses. While single-stranded library preparation (ssLib) has been shown to outperform double-stranded (dsLib) for highly degraded vertebrate host DNA, especially for samples older than 40,000 years, few studies have examined how library protocols shape ancient microbial community reconstruction. Here, we compare the sequencing output of paired ssLib and dsLib dental calculus libraries generated from 12 Neanderthals and two Chalcolithic humans, prepared using implementations of the Gansauge et al. and Meyer and Kircher protocols, respectively, and sequenced with identical Illumina chemistry. We compared read length and GC%, read duplication and taxonomic profiles across normalization strategies to assess protocol-specific biases. Double-stranded libraries retained a significantly higher proportion of sequenced reads throughout data processing (dsLib 72.1%, ssLib 37.9%), a higher proportion of oral reads (dsLib 9.78%, ssLib 6.75%), significantly longer median oral DNA read lengths (dsLib 57.5 bp, ssLib 50.5 bp) and more GC-rich fragments (dsLib 60.5% GC, ssLib 52.5% GC). In contrast, ssLibs exhibited slightly higher Shannon diversity and a greater proportion of unique reads. Despite these differences, species richness and overall community composition was not significantly different between protocols, with individual and preservation status explaining the most variance. Stratifying reads by length (< 50 bp vs. ≥ 50 bp) resulted in different classification rates but only had minor effects on diversity estimates. Together, these results demonstrate that dsLib and ssLib protocols impose distinct trade-offs and library choice should be guided by study-specific goals.
(© 2026 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)
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Grant Information: Werner Siemens-Stiftung; 390713860 Deutsche Forschungsgemeinschaft; National Science Foundation
Contributed Indexing: Keywords: ancient DNA; dental calculus; metagenomics; oral microbiome
Substance Nomenclature: 0 (DNA, Ancient)
0 (DNA, Single-Stranded)
Entry Date(s): Date Created: 20260921 Date Completed: 20260921 Latest Revision: 20260924
Update Code: 20260924
PubMed Central ID: PMC13593575
DOI: 10.1111/1755-0998.70201
PMID: 42767975
Βάση Δεδομένων: MEDLINE
Περιγραφή
ISSN:1755-0998
DOI:10.1111/1755-0998.70201