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ToolsBedarf, Janis R., Beraza, Naiara, Khazneh, Hassan, Özkurt, Ezgi, Baker, David, Borger, Valeri, Wüllner, Ullrich and Hildebrand, Falk (2021) Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals. Microbiome, 9. ISSN 2049-2618
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Abstract
Background: Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods: Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results: Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions: Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains. [MediaObject not available: see fulltext.]
| Item Type: | Article |
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| Additional Information: | Availability of data and materials: The datasets generated and analyzed as part of the current study are publicly available in the ENA repository (https://www.ebi.ac.uk/ena) under the accession number PRJEB42409. Author’s note: as the dataset contains a large proportion of human genomic information, human genomic sequences were removed from the publicly available dataset; the whole dataset including human genomic sequences is available in the EGA repository upon author’s request (https://www.ebi.ac.uk/ega) under the accession number EGAS00001004757. R analysis scripts are available on https://github.com/hildebra/brainMicrobiomeRscripts. Changes to the LotuS pipeline to reliably detect off-targets are implemented in LotuS2 at http://lotus2.earlham.ac.uk/. Funding Information: The authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC); this research was funded by the ParkinsonFonds Deutschland gGmbH and the BBSRC Institute Strategic Programme Gut Microbes and Health BB/R012490/1 and its constituent project(s) BBS/E/F/000PR10353. |
| Uncontrolled Keywords: | 16s rrna gene,bacterial infection,brain,brain-microbiome,microbiome,next generation sequencing,qpcr,microbiology,microbiology (medical) ,/dk/atira/pure/subjectarea/asjc/2400/2404 |
| Faculty \ School: | Faculty of Science > School of Biological Sciences |
| UEA Research Groups: | Faculty of Medicine and Health Sciences > Research Centres > Norwich Institute for Healthy Aging |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 16 Jan 2025 01:10 |
| Last Modified: | 19 Jan 2025 01:00 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/98228 |
| DOI: | 10.1186/s40168-021-01012-1 |
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