Researchers find potential serious problem with sequencing techniques.
DNA contamination is ubiquitous in laboratory equipment commonly used to analyse the microbes that inhabit the human body, potentially seriously undermining undermining cutting-edge work to understand this ‘microbiome’.
The micro-organisms that inhabit the human body have long been known to play a crucial role, but the increasing ease of genetic sequencing has revolutionised this area of study. But many newcomers to the field may be mistaking contamination for breakthroughs, warns Alan Walker, a microbiologist at the University of Aberdeen, UK.
“If you look back in the literature, there’s been stuff about contamination in reagents going back decades. But, he adds, “As microbiota research has boomed, there have been a lot of people drawn into the field who aren’t aware of this literature.”
In one of the first systematic investigations of the problem, Walker and his colleagues show that two commonly used techniques can lead to genuine results being “effectively swamped” by contamination. The team sequenced a pure culture of Salmonella bongori and a series of ten-fold dilutions of this, using standard laboratory practice in three different institutes. Contamination slowly increased through the dilutions, and quickly swamped the original Salmonella signal. They published their results in BMC Biology on 12 November1.
The team traced at least part of the contamination to off-the-shelf kits sold to assist researchers in DNA extraction. The kits are not sold as being sterile, and it is researchers’ fault for assuming that they are, Walker points out.
He told Nature that people working with samples that contain a large number of microbes — such as faecal samples — will probably not have problems, as the original signal is strong enough to overcome the contamination. But microbiome researchers are increasingly pushing into new areas where they are working with ‘low biomass samples’, including spinal fluid, blood, and lungs.
Researchers in Walker’s lab routinely run negative controls alongside the samples they are actually sequencing, to detect contamination. He says that they have detected strains of microbes cropping up in their negative controls that other studies have reported as actual findings.
“We haven’t challenged anyone directly,” he says. “We hope the message will stand for itself. We’re trying to nip it in the bud now before it becomes more of an issue.”
The paper adds to concerns in the scientific community that rapid development of sequencing technology has in some cases outpaced scientists’ ability to use it. In a paper published last month in PLOS ONE, researcher Richard Lusk of the University of Michigan, Ann Arbor, analysed data from four different independent sequencing experiments for potential contamination. He found DNA from a wide variety of different species, with dilute samples more likely to suffer problems.
“Both my paper and this paper came to essentially the same conclusion,” Lusk says, namely, the need to run blank samples alongside an experiment. “However, if that sample is really dilute, and the species you think might be a contaminant is infrequent, that's always going to be really hard,” says Lusk.
He adds that it is especially useful that Walker and colleagues were able to trace so much variation to the DNA extraction kits, which should help researchers design better experiments in future.
Stratec, a biomedical company in Birkenfeld, Germany, which manufactures one of the kits in question said in a statement that their kit was designed for use with stool samples, not from diluted bacterial cultures as in the new study.
The company further says, “The PSP Spin Stool DNA Plus kit is neither marketed as sterile nor as DNA free. Therefore, a certain degree of contaminating material cannot be excluded. Any contamination may indeed be detected through off-label-use application as in the present study.”
The manufacturer of another of the kits analysed in the study, QIAGEN of Hilden, Germany, also stressed that the kit tested was “not designed to be DNA-free or to be used in low-biomass applications”. In a statement the company added, “We welcome this research and believe that it will help to drive awareness of the importance of additional experimental controls in deep sequencing and low-biomass applications.”
Stratec adds that they also recommend the use of negative and other experimental controls to deal with potential contamination. The company says that it has looked into producing DNA-free kits, and concluded that while this is technically feasible, such kits would probably be too expensive to produce.
In the meantime, the warning from experts in the field is caveat experimenter.
“This is a problem that is known in the field for quite some time,” Peer Bork, head of bioinformatics at the European Molecular Biology Laboratory in Heidelberg, Germany, told Nature in an email. “It’s only a part of a much bigger problem as it is not only the kits, but also sampling, DNA extraction in general, library preparation, sequencing, processing et cetera.”
Salter, S. J. et al. BMC Biology 12, 87 (2014).
Lusk, R. W. PLOS ONE 9, e110808 (2014).
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Cressey, D. Contamination threatens microbiome science. Nature (2014). https://doi.org/10.1038/nature.2014.16327