{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T19:40:07Z","timestamp":1792870807636,"version":"4.5.0"},"reference-count":44,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,12,21]],"date-time":"2022-12-21T00:00:00Z","timestamp":1671580800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100005416","name":"Norwegian Research Council","doi-asserted-by":"publisher","award":["273833"],"award-info":[{"award-number":["273833"]}],"id":[{"id":"10.13039\/501100005416","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure\u2122). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis\u2122 showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmicb.2022.1038120","type":"journal-article","created":{"date-parts":[[2022,12,21]],"date-time":"2022-12-21T07:42:00Z","timestamp":1671608520000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":34,"title":["Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies"],"prefix":"10.3389","volume":"13","author":[{"given":"Polona","family":"Rajar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Achal","family":"Dhariwal","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriela","family":"Salvadori","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roger","family":"Junges","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heidi Aar\u00f8","family":"\u00c5mdal","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dag","family":"Berild","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Drude","family":"Fugelseth","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ola Didrik","family":"Saugstad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulrik","family":"Lausten-Thomsen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gorm","family":"Greisen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kirsti","family":"Haaland","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fernanda Cristina","family":"Petersen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,12,21]]},"reference":[{"key":"ref1","article-title":"Fast QC: a quality control tool for high throughput sequence data","author":"Andrews","year":"2010"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"e65088","DOI":"10.7554\/eLife.65088","article-title":"Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bio bakery 3","volume":"10","author":"Beghini","year":"2021","journal-title":"elife"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"e32942","DOI":"10.1371\/journal.pone.0032942","article-title":"Deep sequencing analyses of low density microbial communities: working at the boundary of accurate microbiota detection","volume":"7","author":"Biesbroek","year":"2012","journal-title":"PloS One"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"1283","DOI":"10.1164\/rccm.201407-1240OC","article-title":"Early respiratory microbiota composition determines bacterial succession patterns and respiratory health in children","volume":"190","author":"Biesbroek","year":"2014","journal-title":"Am. 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