According to the paper, published in BMC Biology, many studies investigating the microbiome are likely to have been contaminated and may incorrectly report the presence of microorganisms unintentionally introduced from the laboratory environment
The findings could explain why unexpected bacteria have been previously identified in clinical samples and suggests that pervious research in to the microbiota could have prematurely proposed links to disease.
Led by scientists at the Wellcome Trust Sanger Institute, the researcher systematically shows that contaminating bacteria are easily introduced from DNA extraction kits, chemical reagents and the laboratory environment, and can affect the results of microbiome analyses.
"Recent advances in DNA sequencing technology - which allow extraordinary depth of sequencing - are being used by researchers to analyse extremely sparse microbial populations,” explained lead researcher Dr Alan Walker, now based at the University of Aberdeen. “What we have now shown is that these types of samples are susceptible to contaminant DNA from any source, whether it is at the time of collection, from handling samples, or during the extraction or amplification process.”
“This can critically impact study results, and we're now advising caution to researchers studying microbiota in low biomass environments."
Sequencing science
While the issue of contamination has been reported in the past, for the first time the researchers systematically assessed its impact on samples of varying biomass.
To do this, they took a pure culture of Salmonella bongori bacteria, and investigated the communities of bacteria which were detected following different dilutions of the culture.
Not only did they see an increase in the abundance of contaminating bacterial DNA with increasing dilutions, but at the highest dilutions, the amount of contaminating DNA even exceeded the original Salmonella DNA, reported the team.
The study was repeated across different laboratories, and the contaminating bacterial varieties were found to differ between locations, due to the contaminants present in different chemical reagents and the wider environment.
Biomass sample
In some cases, Walker and his colleagues found that 270 different varieties of bacteria were present in control samples, when only one variety would have been observed if there was no contamination at all.
Many of the contaminating varieties of bacteria were those normally found in soil and water, and on the human skin, said the team – who also noted a great deal of overlap between the varieties of contaminating bacteria found in their study, and those that have been reported as noteworthy in previous low biomass microbiome studies.
Indeed, the Sanger Institute team suggested that samples containing low amounts of material or 'biomass', such as those taken from the blood or lungs, are far more likely to be prone to contamination than high-biomass samples from faeces.
False diversity?
To show how contamination could have further impacts on studies, the team also investigated nasal swab samples from a Thai-Burmese refugee camp, which have previously been used to study the carriage of pneumococcal disease.
The researchers initially observed patterns of nasal microbiota variation, but later realised that these were simply due to different types of contamination from the various batches of DNA extraction kits.
Walker and colleagues said the findings serve as a reminder of the significant, and potentially misleading, impact that contaminants can have on microbiome studies and their conclusions.
To address these contamination issues, they suggested a number of steps which researchers can take when studying low biomass samples. These include using negative controls to identify varieties of bacteria that might be contaminating samples, maximizing the biomass of the starting sample through filtration or enrichment, and minimizing the risk of contamination during sample collection.
Source: BMC Biology
Published online ahead of print, doi: 10.1186/s12915-014-0087-z
“Reagent and laboratory contamination can critically impact sequence-based microbiome analyses”
Authors: Susannah J Salter, Michael J Cox, et al