Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

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.

A closer look at the mycobiome in Alzheimer's disease: Fungal species, pathogenesis and transmission

“…However, besides AD and PD, fungi species were not detected in the control samples in other neurodegenerative disease comparative studies such as multiple sclerosis (Alonso, Fernández‐Fernández, et al, 2018) and amyotrophic lateral sclerosis (Alonso, Pisa, Fernández‐Fernández, et al, 2017). A recent study done by Bedarf et al (2021) demonstrated that the bacterial signals detected in human brains was due to a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%) using a low biomass 16S rRNA gene sequencing. These were due to the off‐target amplification in low‐bacterial/high‐host DNA samples, where the amplified DNA sequences were clustered and falsely assigned to bacterial taxa (Bedarf et al, 2021).…”

“…A recent study done by Bedarf et al (2021) demonstrated that the bacterial signals detected in human brains was due to a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%) using a low biomass 16S rRNA gene sequencing. These were due to the off‐target amplification in low‐bacterial/high‐host DNA samples, where the amplified DNA sequences were clustered and falsely assigned to bacterial taxa (Bedarf et al, 2021). By including a suitable negative and positive controls to rule out pre‐ and post‐sequencing contamination, there was no true taxonomic signal that could confirm the presence of microbes in either healthy or brains from PD patients (Bedarf et al, 2021).…”

Reconstitution and Transmission of Gut Microbiomes and Their Genes between Generations

“…Maqsood et al [264] reported that in his study several infant stool bacterial microbiomes were indistinguishable from buffer background, and thus cannot be attributed to maternal transmission. In a different study, 54% of bacterial signals in the brain was explained by exogenous DNA contamination, and were thus falsepositives [265]. DNase treatment has been suggested as a method for removing DNA contamination in PCR reagents [263].…”

LotuS2: An ultrafast and highly accurate tool for amplicon sequencing analysis

“…OTUs/ASVs are by default aligned against the phiX genome, a synthetic genome often included in Illumina sequencing runs, using Minimap2 [33]; these OTUs/ASVs are subsequently removed. Additionally, the user can filter for host contamination by providing custom genomes (e.g., human reference), as host genome reads are often misclassified as bacterial 16S by existing pipelines [3].…”

“…Fungi), respectively [1,2]. The popularity of amplicon sequencing has been growing due to its broad applicability, ease-of-use, costefficiency, streamlined analysis workflows as well as specialist applications such as low biomass sampling [3].…”