Reagent contamination can critically impact sequence-based microbiome analyses

The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR based 16S rRNA gene surveys and shotgun metagenomics. These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. Concurrent sequencing of negative control samples is strongly advised.

[1]  E. Tóth,et al.  Bacterial communities in an ultrapure water containing storage tank of a power plant. , 2011, Acta microbiologica et immunologica Hungarica.

[2]  A. Andremont,et al.  Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes , 1997, Molecular biotechnology.

[3]  Hua Shen,et al.  Sensitive, real-time PCR detects low-levels of contamination by Legionella pneumophila in commercial reagents. , 2006, Molecular and cellular probes.

[4]  Norman R. Pace,et al.  Specific Ribosomal DNA Sequences from Diverse Environmental Settings Correlate with Experimental Contaminants , 1998, Applied and Environmental Microbiology.

[5]  N. Taylor,et al.  DNA extraction from low-biomass carbonate rock: an improved method with reduced contamination and the low-biomass contaminant database. , 2006, Journal of microbiological methods.

[6]  M. von Knebel Doeberitz,et al.  Characterization of contaminating DNA in Taq polymerase which occurs during amplification with a primer set for Legionella 5S ribosomal RNA. , 1994, Molecular and cellular probes.

[7]  J. Shamonki,et al.  Microbial Dysbiosis Is Associated with Human Breast Cancer , 2014, PloS one.

[8]  K. Konstantinidis,et al.  Microbiome of the upper troposphere: Species composition and prevalence, effects of tropical storms, and atmospheric implications , 2012, Proceedings of the National Academy of Sciences.

[9]  E. Stackebrandt,et al.  Nucleic acid techniques in bacterial systematics , 1991 .

[10]  N. Zou,et al.  Presence of Bacterial Phage-Like DNA Sequences in Commercial Taq DNA Polymerase Reagents , 2004, Journal of Clinical Microbiology.

[11]  C. Roberts,et al.  Using ancient DNA analysis in palaeopathology: a critical analysis of published papers, with recommendations for future work , 2008 .

[12]  E. Tóth,et al.  Application of special oligotrophic media for cultivation of bacterial communities originated from ultrapure water. , 2013, Acta microbiologica et immunologica Hungarica.

[13]  M. Surette,et al.  Brain Microbial Populations in HIV/AIDS: α-Proteobacteria Predominate Independent of Host Immune Status , 2013, PloS one.

[14]  V. Kunin,et al.  Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. , 2009, Environmental microbiology.

[15]  U. Göbel,et al.  Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. , 1997, FEMS microbiology reviews.

[16]  Patrick D. Schloss,et al.  Reducing the Effects of PCR Amplification and Sequencing Artifacts on 16S rRNA-Based Studies , 2011, PloS one.

[17]  M. Frith,et al.  Adaptive seeds tame genomic sequence comparison. , 2011, Genome research.

[18]  D. Labuda,et al.  Use of gamma irradiation to eliminate DNA contamination for PCR. , 1990, Nucleic acids research.

[19]  Martin Hartmann,et al.  Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities , 2009, Applied and Environmental Microbiology.

[20]  Ram Veerapaneni,et al.  Microbial Analyses of Ancient Ice Core Sections from Greenland and Antarctica , 2013, Biology.

[21]  D. Antonopoulos,et al.  Diversity of bacteria at healthy human conjunctiva. , 2011, Investigative ophthalmology & visual science.

[22]  K. Nelson,et al.  Reagent decontamination to eliminate false-positives in Escherichia coli qPCR. , 2008, Journal of microbiological methods.

[23]  Thomas Bonfert,et al.  Mining RNA–Seq Data for Infections and Contaminations , 2013, PloS one.

[24]  T. Pitt,et al.  Development of an ethidium monoazide–enhanced internally controlled universal 16S rDNA real‐time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates , 2012, Transfusion.

[25]  T. Torres,et al.  Bacterial communities and species-specific associations with the mucus of Brazilian coral species , 2013, Scientific Reports.

[26]  F. Graham,et al.  Methods for construction of adenovirus vectors , 1995, Molecular biotechnology.

[27]  S. Klaschik,et al.  Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR , 2002, Molecular biotechnology.

[28]  M. Meyerson,et al.  Sequence-based discovery of Bradyrhizobium enterica in cord colitis syndrome. , 2013, The New England journal of medicine.

[29]  A. Mira,et al.  Detection of Transient Bacteraemia following Dental Extractions by 16S rDNA Pyrosequencing: A Pilot Study , 2013, PloS one.

[30]  H. Shahbazkia,et al.  Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection , 2012, PLoS pathogens.

[31]  D. Labuda,et al.  Use of γ irradiation to eliminate DNA contamination for PCR , 1990 .

[32]  S. James,et al.  A global survey of the bacteria within earthworm nephridia. , 2013, Molecular phylogenetics and evolution.

[33]  Nansheng Chen,et al.  Metagenomic analysis of the pinewood nematode microbiome reveals a symbiotic relationship critical for xenobiotics degradation , 2013, Scientific Reports.

[34]  Christine Wendt,et al.  Longitudinal analysis of the lung microbiome in lung transplantation. , 2013, FEMS microbiology letters.

[35]  Mechthild Prinz,et al.  The Application of Ultraviolet Irradiation to Exogenous Sources of DNA in Plasticware and Water for the Amplification of Low Copy Number DNA , 2006, Journal of Forensic Sciences.

[36]  R. Borrow,et al.  Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR , 2000, Journal of Clinical Microbiology.

[37]  K. Botzenhart,et al.  DNase Pretreatment of Master Mix Reagents Improves the Validity of Universal 16S rRNA Gene PCR Results , 2003, Journal of Clinical Microbiology.

[38]  K. Rand,et al.  Taq polymerase contains bacterial DNA of unknown origin. , 1990, Molecular and cellular probes.

[39]  C. Tseng,et al.  An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents , 2011, PloS one.

[40]  J. Doré,et al.  Involvement of tissue bacteria in the onset of diabetes in humans: evidence for a concept , 2011, Diabetologia.

[41]  Mihai Pop,et al.  Statistical Methods for Detecting Differentially Abundant Features in Clinical Metagenomic Samples , 2009, PLoS Comput. Biol..

[42]  H. Morgan,et al.  Removal of contaminating DNA from polymerase chain reaction using ethidium monoazide. , 2007, Journal of microbiological methods.

[43]  Thierry Grange,et al.  An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications , 2010, PloS one.

[44]  G. Srinivas,et al.  Genome-wide mapping of gene–microbiota interactions in susceptibility to autoimmune skin blistering , 2013, Nature Communications.

[45]  Russell J. Davenport,et al.  Removing Noise From Pyrosequenced Amplicons , 2011, BMC Bioinformatics.

[46]  S. Schuster,et al.  Integrative analysis of environmental sequences using MEGAN4. , 2011, Genome research.

[47]  Julian Parkhill,et al.  Patent Human Infections with the Whipworm, Trichuris trichiura, Are Not Associated with Alterations in the Faecal Microbiota , 2013, PloS one.

[48]  K. Ogden,et al.  Survival and nutritional requirements of three bacteria isolated from ultrapure water , 2002, Journal of Industrial Microbiology and Biotechnology.

[49]  H. Poinar,et al.  Ancient DNA: Do It Right or Not at All , 2000, Science.

[50]  P. Morris,et al.  Subglacial Lake Vostok (Antarctica) Accretion Ice Contains a Diverse Set of Sequences from Aquatic, Marine and Sediment-Inhabiting Bacteria and Eukarya , 2013, PloS one.

[51]  J. O’Hanlon,et al.  Analysis of Bacteria Contaminating Ultrapure Water in Industrial Systems , 2002, Applied and Environmental Microbiology.

[52]  A. Klindworth,et al.  Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies , 2012, Nucleic acids research.

[53]  S. Hughes,et al.  Multiple Sources of Contamination in Samples from Patients Reported to Have XMRV Infection , 2012, PloS one.

[54]  Kasthuri Venkateswaran,et al.  New perspectives on viable microbial communities in low-biomass cleanroom environments , 2012, The ISME Journal.

[55]  K. Nelson,et al.  Pyrosequencing analysis of the human microbiota of healthy Chinese undergraduates , 2013, BMC Genomics.

[56]  G. Sarkar,et al.  Shedding light on PCR contamination , 1990, Nature.

[57]  Gabriele Berg,et al.  The ignored diversity: complex bacterial communities in intensive care units revealed by 16S pyrosequencing , 2013, Scientific Reports.

[58]  E. Willerslev,et al.  Isolation of nucleic acids and cultures from fossil ice and permafrost. , 2004, Trends in ecology & evolution.

[59]  J. Jonasson,et al.  Identification of mixed bacterial DNA contamination in broad-range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons. , 2003, FEMS microbiology letters.

[60]  F. Nosten,et al.  A Longitudinal Study of Streptococcus pneumoniae Carriage in a Cohort of Infants and Their Mothers on the Thailand-Myanmar Border , 2012, PloS one.

[61]  Sarah L. Westcott,et al.  Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform , 2013, Applied and Environmental Microbiology.

[62]  C. Vandenbroucke-Grauls,et al.  Optimization of Real-Time PCR Assay for Rapid and Sensitive Detection of Eubacterial 16S Ribosomal DNA in Platelet Concentrates , 2003, Journal of Clinical Microbiology.

[63]  Björn Usadel,et al.  Trimmomatic: a flexible trimmer for Illumina sequence data , 2014, Bioinform..

[64]  Gregory J. Phillips,et al.  Bacterial Community Profiling of Milk Samples as a Means to Understand Culture-Negative Bovine Clinical Mastitis , 2013, PloS one.

[65]  C. Vandenbroucke-Grauls,et al.  Removal of contaminating DNA from commercial nucleic acid extraction kit reagents. , 2005, Journal of microbiological methods.

[66]  R. Skuce,et al.  Identification and elimination of DNA sequences in Taq DNA polymerase , 1994, Journal of clinical microbiology.

[67]  Rohan S. Kulkarni,et al.  Enrichment of lung microbiome with supraglottic taxa is associated with increased pulmonary inflammation , 2013, Microbiome.

[68]  G. McFeters,et al.  Distribution of bacteria within operating laboratory water purification systems , 1993, Applied and environmental microbiology.

[69]  K. Venkateswaran,et al.  Microbial Monitoring of Spacecraft and Associated Environments , 2004, Microbial Ecology.

[70]  Douglas E. Brash,et al.  Common Contaminants in Next-Generation Sequencing That Hinder Discovery of Low-Abundance Microbes , 2014, PloS one.

[71]  N. Carroll,et al.  Elimination of Bacterial DNA from TaqDNA Polymerases by Restriction Endonuclease Digestion , 1999, Journal of Clinical Microbiology.