{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,26]],"date-time":"2026-10-26T13:33:25Z","timestamp":1793021605922,"version":"4.5.0"},"reference-count":111,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T00:00:00Z","timestamp":1789948800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T00:00:00Z","timestamp":1789948800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["390713860"],"award-info":[{"award-number":["390713860"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Molecular Ecology Resources"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>DNA library construction methods can affect the recovery of ancient DNA, thus influencing downstream analyses. While single\u2010stranded library preparation (ssLib) has been shown to outperform double\u2010stranded (dsLib) for highly degraded vertebrate host DNA, especially for samples older than 40,000\u2009years, 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\u00a0al. 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\u2010specific biases. Double\u2010stranded 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\u2009bp, ssLib 50.5\u2009bp) and more GC\u2010rich 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;\u200950\u2009bp vs. \u2265\u200950\u2009bp) 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\u2010offs and library choice should be guided by study\u2010specific goals.<\/jats:p>","DOI":"10.1111\/1755-0998.70201","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T02:36:21Z","timestamp":1790044581000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparing the Performance of Double\u2010Stranded and Single\u2010Stranded\n                    <scp>DNA<\/scp>\n                    Libraries for Ancient Oral Microbiome Reconstruction"],"prefix":"10.1111","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8359-4988","authenticated-orcid":false,"given":"Keri","family":"Burge","sequence":"first","affiliation":[{"name":"Department of Anthropology Harvard University  Cambridge Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9810-9917","authenticated-orcid":false,"given":"Irina M.","family":"Velsko","sequence":"additional","affiliation":[{"name":"Department of Archaeogenetics Max Planck Institute for Evolutionary Anthropology  Leipzig Germany"},{"name":"Associated Research Group of Archaeogenetics Leibniz Institute for Natural Product Research and Infection Biology, Hans Kn\u00f6ll Institute  Jena Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8017-0194","authenticated-orcid":false,"given":"Domingo C.","family":"Salazar\u2010Garc\u00eda","sequence":"additional","affiliation":[{"name":"Departament de Prehist\u00f2ria, Arqueologia i Hist\u00f2ria Antiga Universitat de Val\u00e8ncia  Val\u00e8ncia Spain"},{"name":"Department of Geological Sciences University of Cape Town  Cape Town South\u00a0Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4686-5424","authenticated-orcid":false,"given":"Mar\u00eda Haber","family":"Uriarte","sequence":"additional","affiliation":[{"name":"Departamento de Prehistoria, Arqueolog\u00eda, Historia Antigua, Historia Medieval y Ciencias y T\u00e9cnicas Historiogr\u00e1ficas Universidad de Murcia  Murcia Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0874-6213","authenticated-orcid":false,"given":"Joaqu\u00edn","family":"Lomba Maurandi","sequence":"additional","affiliation":[{"name":"Departamento de Prehistoria, Arqueolog\u00eda, Historia Antigua, Historia Medieval y Ciencias y T\u00e9cnicas Historiogr\u00e1ficas Universidad de Murcia  Murcia Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4528-5877","authenticated-orcid":false,"given":"Christina","family":"Warinner","sequence":"additional","affiliation":[{"name":"Department of Anthropology Harvard University  Cambridge Massachusetts USA"},{"name":"Department of Archaeogenetics Max Planck Institute for Evolutionary Anthropology  Leipzig Germany"},{"name":"Associated Research Group of Archaeogenetics Leibniz Institute for Natural Product Research and Infection Biology, Hans Kn\u00f6ll Institute  Jena Germany"},{"name":"Faculty of Biological Sciences Friedrich Schiller University  Jena Germany"},{"name":"Department of Human Evolutionary Biology Harvard University  Cambridge Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,21]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1111\/prd.12151"},{"key":"e_1_2_12_3_1","volume-title":"FastQC: A Quality Control Tool for High Throughput Sequence Data","author":"Andrews S.","year":"2010"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/ajpa.23111"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.17504\/protocols.io.bidyka7w"},{"key":"e_1_2_12_6_1","doi-asserted-by":"crossref","unstructured":"Aron F. 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