Academic Journal
Comparing the Performance of Double-Stranded and Single-Stranded DNA Libraries for Ancient Oral Microbiome Reconstruction.
| 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 |
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| Items | – Name: Title Label: Title Group: Ti Data: Comparing the Performance of Double-Stranded and Single-Stranded DNA Libraries for Ancient Oral Microbiome Reconstruction. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AU" term="%22Burge+K%22">Burge K</searchLink>; Department of Anthropology, Harvard University, Cambridge, Massachusetts, USA.<br /><searchLink fieldCode="AU" term="%22Velsko+IM%22">Velsko IM</searchLink>; 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.<br /><searchLink fieldCode="AU" term="%22Salazar-García+DC%22">Salazar-García DC</searchLink>; Departament de Prehistòria, Arqueologia i Història Antiga, Universitat de València, València, Spain.; Department of Geological Sciences, University of Cape Town, Cape Town, South Africa.<br /><searchLink fieldCode="AU" term="%22Uriarte+MH%22">Uriarte MH</searchLink>; Departamento de Prehistoria, Arqueología, Historia Antigua, Historia Medieval y Ciencias y Técnicas Historiográficas, Universidad de Murcia, Murcia, Spain.<br /><searchLink fieldCode="AU" term="%22Lomba+Maurandi+J%22">Lomba Maurandi J</searchLink>; Departamento de Prehistoria, Arqueología, Historia Antigua, Historia Medieval y Ciencias y Técnicas Historiográficas, Universidad de Murcia, Murcia, Spain.<br /><searchLink fieldCode="AU" term="%22Warinner+C%22">Warinner C</searchLink>; 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. – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22101465604%22">Molecular ecology resources</searchLink> [Mol Ecol Resour] 2026 Oct; Vol. 26 (7), pp. e70201. – Name: TypePub Label: Publication Type Group: TypPub Data: Journal Article; Comparative Study; Evaluation Study – Name: Language Label: Language Group: Lang Data: English – Name: TitleSource Label: Journal Info Group: Src Data: <i>Publisher: </i><searchLink fieldCode="PB" term="%22Blackwell%22">Blackwell </searchLink><i>Country of Publication: </i>England <i>NLM ID: </i>101465604 <i>Publication Model: </i>Print <i>Cited Medium: </i>Internet <i>ISSN: </i>1755-0998 (Electronic) <i>Linking ISSN: </i><searchLink fieldCode="IS" term="%221755098X%22">1755098X </searchLink><i>NLM ISO Abbreviation: </i>Mol Ecol Resour <i>Subsets: </i>MEDLINE – Name: PublisherInfo Label: Imprint Name(s) Group: PubInfo Data: <i>Original Publication</i>: Oxford, England : Blackwell – Name: SubjectMESH Label: MeSH Terms Group: Su Data: <searchLink fieldCode="MM" term="%22Mouth%22">Mouth*</searchLink>/<searchLink fieldCode="MM" term="%22Mouth+microbiology%22">microbiology</searchLink> <br /><searchLink fieldCode="MM" term="%22Metagenomics%22">Metagenomics*</searchLink>/<searchLink fieldCode="MM" term="%22Metagenomics+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Microbiota%22">Microbiota*</searchLink> <br /><searchLink fieldCode="MM" term="%22Gene+Library%22">Gene Library*</searchLink> <br /><searchLink fieldCode="MM" term="%22DNA%2C+Ancient%22">DNA, Ancient*</searchLink><br /><searchLink fieldCode="MH" term="%22DNA%2C+Single-Stranded%22">DNA, Single-Stranded</searchLink>/<searchLink fieldCode="MH" term="%22DNA%2C+Single-Stranded+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Animals%22">Animals</searchLink> ; <searchLink fieldCode="MH" term="%22Humans%22">Humans</searchLink> ; <searchLink fieldCode="MH" term="%22Sequence+Analysis%2C+DNA%22">Sequence Analysis, DNA</searchLink> ; <searchLink fieldCode="MH" term="%22Neanderthals%22">Neanderthals</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: 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 (&lt; 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.<br /> (© 2026 The Author(s). 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(PMID: <searchLink fieldCode="PM" term="%2231182199%22">31182199)</searchLink> – Name: GrantInfo Label: Grant Information Group: Grant Data: Werner Siemens-Stiftung; 390713860 Deutsche Forschungsgemeinschaft; National Science Foundation – Name: SubjectMinor Label: Contributed Indexing Group: Data: <i>Keywords: </i>ancient DNA; dental calculus; metagenomics; oral microbiome – Name: NumberCAS Label: Substance Nomenclature Group: ID Data: 0 (DNA, Ancient)<br />0 (DNA, Single-Stranded) – Name: DateEntry Label: Entry Date(s) Group: Date Data: <i>Date Created: </i>20260921 <i>Date Completed: </i>20260921 <i>Latest Revision: </i>20260924 – Name: DateUpdate Label: Update Code Group: Date Data: 20260924 – Name: PubmedCentralID Label: PubMed Central ID Group: ID Data: PMC13593575 – Name: DOI Label: DOI Group: ID Data: 10.1111/1755-0998.70201 – Name: AN Label: PMID Group: ID Data: 42767975 |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1111/1755-0998.70201 Languages: – Code: eng Text: English PhysicalDescription: Pagination: StartPage: e70201 Subjects: – SubjectFull: DNA, Single-Stranded genetics Type: general – SubjectFull: Animals Type: general – SubjectFull: Humans Type: general – SubjectFull: Sequence Analysis, DNA Type: general – SubjectFull: Neanderthals Type: general – SubjectFull: Mouth microbiology Type: general – SubjectFull: Metagenomics methods Type: general – SubjectFull: Microbiota Type: general – SubjectFull: Gene Library Type: general – SubjectFull: DNA, Ancient Type: general Titles: – TitleFull: Comparing the Performance of Double-Stranded and Single-Stranded DNA Libraries for Ancient Oral Microbiome Reconstruction. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Burge K – PersonEntity: Name: NameFull: Velsko IM – PersonEntity: Name: NameFull: Salazar-García DC – PersonEntity: Name: NameFull: Uriarte MH – PersonEntity: Name: NameFull: Lomba Maurandi J – PersonEntity: Name: NameFull: Warinner C IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 10 Text: 2026 Oct Type: published Y: 2026 Identifiers: – Type: issn-electronic Value: 1755-0998 Numbering: – Type: volume Value: 26 – Type: issue Value: 7 Titles: – TitleFull: Molecular ecology resources Type: main |
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