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dc.contributor.authorDrengenes, Christine
dc.contributor.authorEagan, Tomas Mikal
dc.contributor.authorHaaland, Ingvild
dc.contributor.authorWiker, Harald G
dc.contributor.authorNielsen, Rune
dc.date.accessioned2021-08-09T12:48:57Z
dc.date.available2021-08-09T12:48:57Z
dc.date.created2021-01-20T21:50:10Z
dc.date.issued2021
dc.identifier.issn1471-2164
dc.identifier.urihttps://hdl.handle.net/11250/2767045
dc.description.abstractBackground Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.en_US
dc.language.isoengen_US
dc.publisherBMCen_US
dc.rightsNavngivelse 4.0 Internasjonal*
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/deed.no*
dc.titleExploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4en_US
dc.typeJournal articleen_US
dc.typePeer revieweden_US
dc.description.versionpublishedVersionen_US
dc.rights.holderCopyright 2021 The Authorsen_US
dc.source.articlenumber3en_US
cristin.ispublishedtrue
cristin.fulltextoriginal
cristin.qualitycode1
dc.identifier.doi10.1186/s12864-020-07252-z
dc.identifier.cristin1876092
dc.source.journalBMC Genomicsen_US
dc.identifier.citationBMC Genomics. 2021, 22, 3en_US
dc.source.volume22en_US


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