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BMC Microbiology

, 16:73

Microbial genetics, genomics and proteomics

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.

KeywordsContaminant DNA Bacterial communities 16S rRNA gene sequencing Microbiome AbbreviationsNECnegative extraction control

NEC Bnegative extraction control obtained by substituting culture for lysis buffer

NEC Wnegative extraction control obtained by substituting culture for water

NTC Wno-template PCR or qPCR control

OTUoperational taxonomic unit

R-OTUthe ratio between mean ‘absolute’ abundance of OTUs in NEC and culture samples

Electronic supplementary materialThe online version of this article doi:10.1186-s12866-016-0689-4 contains supplementary material, which is available to authorized users.

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Autor: Vladimir Lazarevic - Nadia Gaïa - Myriam Girard - Jacques Schrenzel

Fuente: https://link.springer.com/







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