Abstract
BackgroundIdentification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA. The sources of these contaminants are reagents used in DNA extraction, PCR, and next-generation sequencing library preparation, and human (skin, oral and respiratory) microbiota from the investigators.ResultsFor in silico removal of reagent contaminants, a pipeline was used which combines the relative abundance of operational taxonomic units (OTUs) in V3–4 16S rRNA gene amplicon datasets with bacterial DNA quantification based on qPCR targeting of the V3 segment of the 16S rRNA gene. Serially diluted cultures of Escherichia coli and Staphylococcus aureus were used for 16S rDNA profiling, and DNA from each of these species was used as a qPCR standard. OTUs assigned to Escherichia or Staphylococcus were virtually unaffected by the decontamination procedure, whereas OTUs from Pseudomonas, which is a major reagent contaminant, were completely or nearly completely removed. The decontamination procedure also attenuated the trend of increase in OTU richness in serially diluted cultures.ConclusionsRemoval of contaminant sequences derived from reagents based on use of qPCR data may improve taxonomic representation in samples with low DNA concentration. Using the described pipeline, OTUs derived from cross-contamination of negative extraction controls were not recognized as contaminants and not removed from the sample dataset.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0689-4) contains supplementary material, which is available to authorized users.
Highlights
Identification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA
Bacterial load determined by culture and quantitative real-time PCR (qPCR) Staphylococcus aureus and Escherichia coli overnight cultures were washed and concentrated, and resulted in 3.5x1010 and 5.3x109 colony-forming units (CFU)/ml, respectively
Our pipeline for removal of reagent contaminants in sequence datasets combines the relative abundance of operational taxonomic unit (OTU) in V3–4 16S rRNA gene amplicon datasets with bacterial DNA quantification based on qPCR targeting of the V3 segment of the 16S rRNA gene
Summary
Identification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA. The sources of these contaminants are reagents used in DNA extraction, PCR, and next-generation sequencing library preparation, and human (skin, oral and respiratory) microbiota from the investigators. The development of PCR and next-generation sequencing techniques has facilitated studying microbial communities without it being necessary to culture individual members. Culture-free approaches may introduce biases in the experimental pipeline, starting from DNA extraction through the generation of sequencing libraries to data analysis. An OTU that corresponds to the genuine member of the microbiota of interest may be found in relevant
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