Comparison of four DNA extraction methods for comprehensive assessment of 16S rRNA bacterial diversity in marine biofilms using high-throughput sequencing

High-throughput DNA sequencing technologies are increasingly used for the metagenomic characterisation of microbial biodiversity. However, basic issues, such as the choice of an appropriate DNA extraction method, are still not resolved for non-model microbial communities. This study evaluates four commonly used DNA extraction methods for marine periphyton biofilms in terms of DNA yield, efficiency, purity, integrity and resulting 16S rRNA bacterial diversity. Among the tested methods, the Plant DNAzol® Reagent (PlantDNAzol) and the FastDNA® SPIN Kit for Soil (FastDNA Soil) methods were best suited to extract high quantities of DNA (77-130 μg g wet wt-1). Lower amounts of DNA were obtained (<37 μg g wet wt-1) with the Power Plant® Pro DNA Isolation Kit (PowerPlant) and the Power Biofilm® DNA Isolation Kit (PowerBiofilm) methods, but integrity and purity of the extracted DNA were higher. Results from 16S rRNA amplicon sequencing demonstrate that the choice of a DNA extraction method significantly influences the bacterial community profiles generated. A higher number of bacterial OTUs were detected when DNA was extracted with the PowerBiofilm and the PlantDNAzol methods. Overall, this study demonstrates the potential bias in metagenomic diversity estimates associated with different DNA extraction methods.

[1]  R. Tibshirani,et al.  (37) Medications as Independent Risk Factors of Delirium in Patients With COVID-19: A Retrospective Study , 2018, Journal of the Academy of Consultation-Liaison Psychiatry.

[2]  Karell Martínez Guijarro,et al.  A retrospective analysis of contamination and periphyton PICT patterns for the antifoulant irgarol 1051, around a small marina on the Swedish west coast. , 2009, Marine pollution bulletin.

[3]  Erik Kristiansson,et al.  Statistical evaluation of methods for identification of differentially abundant genes in comparative metagenomics , 2016, BMC Genomics.

[4]  Robert K. Colwell,et al.  A new statistical approach for assessing similarity of species composition with incidence and abundance data , 2004 .

[5]  J. Hollender,et al.  Micropollutant-induced tolerance of in situ periphyton: Establishing causality in wastewater-impacted streams. , 2017, Water research.

[6]  C. Michel,et al.  Representative sampling of natural biofilms: influence of substratum type on the bacterial and fungal communities structure , 2016, SpringerPlus.

[7]  B. Javornik,et al.  Robust CTAB-activated charcoal protocol for plant DNA extraction , 2006, Acta agriculturae Slovenica.

[8]  Mads Albertsen,et al.  Back to Basics – The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities , 2015, PloS one.

[9]  K. Sundbäck,et al.  Benthic nitrogen fluxes along a depth gradient in a microtidal fjord: The role of denitrification and microphytobenthos , 2004 .

[10]  S. Sørensen,et al.  Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis , 2004 .

[11]  Seth Love,et al.  Genetic Evidence Implicates the Immune System and Cholesterol Metabolism in the Aetiology of Alzheimer's Disease , 2010, PloS one.

[12]  V. O’Flaherty,et al.  DNA extraction method affects microbial community profiles from soils and sediment , 2007, Applied Microbiology and Biotechnology.

[13]  M. Manefield,et al.  The roles of extracellular DNA in the structural integrity of extracellular polymeric substance and bacterial biofilm development. , 2013, Environmental microbiology reports.

[14]  P. Dixon.  VEGAN, a package of R functions for community ecology , 2003 .

[15]  K. Stokes,et al.  Marine biofilms on artificial surfaces: structure and dynamics. , 2013, Environmental microbiology.

[16]  T. Royer,et al.  Pharmaceuticals suppress algal growth and microbial respiration and alter bacterial communities in stream biofilms. , 2013, Ecological applications : a publication of the Ecological Society of America.

[17]  W. Nelson,et al.  Comparative Analysis of Eukaryotic Marine Microbial Assemblages from 18S rRNA Gene and Gene Transcript Clone Libraries by Using Different Methods of Extraction , 2012, Applied and Environmental Microbiology.

[18]  M. Esumi,et al.  Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions , 2016, PloS one.

[19]  A. Bull,et al.  Microbial diversity , 2004, Biodiversity & Conservation.

[20]  H. Blanck,et al.  A phylogenetic approach to detect selection on the target site of the antifouling compound irgarol in tolerant periphyton communities. , 2009, Environmental microbiology.

[21]  C. Georgiou,et al.  Assay for the quantification of intact/fragmented genomic DNA. , 2006, Analytical biochemistry.

[22]  D. M. Ward,et al.  16S rRNA sequences reveal numerous uncultured microorganisms in a natural community , 1990, Nature.

[23]  Héctor Corrada Bravo,et al.  Intensity normalization improves color calling in SOLiD sequencing , 2010, Nature Methods.

[24]  Lucas Sinclair,et al.  Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform , 2014, bioRxiv.

[25]  Erik Kristiansson,et al.  ShotgunFunctionalizeR: an R-package for functional comparison of metagenomes , 2009, Bioinform..

[26]  H. Blanck,et al.  Validity of an Ecotoxicological Test System: Short-Term and Long-Term Effects of Arsenate on Marine Periphyton Communities in Laboratory Systems , 1988 .

[27]  Anders F. Andersson,et al.  Pyrosequencing reveals contrasting seasonal dynamics of taxa within Baltic Sea bacterioplankton communities , 2010, The ISME Journal.

[28]  J. Newbold,et al.  Contributions of microbial biofilms to ecosystem processes in stream mesocosms , 2003, Nature.

[29]  A. Clooney,et al.  16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform , 2016, BMC Microbiology.

[30]  T. Stoeck,et al.  Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water , 2010, Molecular ecology.

[31]  X. Timoner,et al.  The dynamics of biofilm bacterial communities is driven by flow wax and wane in a temporary stream , 2014 .

[32]  D. Barceló,et al.  Effects of flow intermittency and pharmaceutical exposure on the structure and metabolism of stream biofilms. , 2015, The Science of the total environment.

[33]  J. Clapp,et al.  PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods. , 2001, FEMS microbiology ecology.

[34]  S. Sabater,et al.  Monitoring the effect of chemicals on biological communities. The biofilm as an interface , 2007, Analytical and bioanalytical chemistry.

[35]  Frédéric Garabetian,et al.  Analysis of bacterial diversity in river biofilms using 16S rDNA PCR-DGGE: methodological settings and fingerprints interpretation. , 2005, Water research.

[36]  H. Bürgmann,et al.  A strategy for optimizing quality and quantity of DNA extracted from soil. , 2001, Journal of microbiological methods.

[37]  M. Tucker,et al.  Extraction of high-quality genomic DNA from latex-containing plants. , 2003, Analytical biochemistry.

[38]  M. Phillips,et al.  Microbial diversity in European alpine permafrost and active layers. , 2016, FEMS microbiology ecology.

[39]  Robert C. Edgar,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2001 .

[40]  D. Allison,et al.  The Biofilm Matrix , 2003, Biofouling.

[41]  J. Humbert,et al.  A high-throughput sequencing ecotoxicology study of freshwater bacterial communities and their responses to tebuconazole. , 2014, FEMS microbiology ecology.

[42]  J. Ravel,et al.  Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome , 2012, PloS one.

[43]  Thikra. A. Abed.  Evaluation of methods for the extraction and purification of DNA of cultured Lactobacillus colony isolated from dairy products , 2013 .

[44]  H. Ochman,et al.  Illumina-based analysis of microbial community diversity , 2011, The ISME Journal.

[45]  Frank Vitzthum,et al.  Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments. , 2003, Nucleic acids research.

[46]  H. E. Tegetmeyer,et al.  Quantification of the effects of ocean acidification on sediment microbial communities in the environment: the importance of ecosystem approaches , 2016, FEMS microbiology ecology.

[47]  A. Felczykowska,et al.  Sampling, metadata and DNA extraction - important steps in metagenomic studies. , 2015, Acta biochimica Polonica.

[48]  Daniel G. Brown,et al.  PANDAseq: paired-end assembler for illumina sequences , 2012, BMC Bioinformatics.

[49]  T. E. Cloete,et al.  Molecular Techniques for Determining Microbial Diversity and Community Structure in Natural Environments , 2000, Critical reviews in microbiology.

[50]  V. Parro,et al.  Environmental transcriptome analysis reveals physiological differences between biofilm and planktonic modes of life of the iron oxidizing bacteria Leptospirillum spp. in their natural microbial community , 2010, BMC Genomics.

[51]  C. Blackwood,et al.  Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil , 2009, Applied and Environmental Microbiology.

[52]  H. Shirakawa,et al.  Serotonin Improves High Fat Diet Induced Obesity in Mice , 2016, PloS one.

[53]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[54]  John R. Lawrence,et al.  Next-generation sequencing of microbial communities in the Athabasca River 1 and its tributaries in relation to oil sands mining activities 2 3 , 2012 .

[55]  D. Barceló,et al.  Pollution-induced community tolerance to non-steroidal anti-inflammatory drugs (NSAIDs) in fluvial biofilm communities affected by WWTP effluents. , 2014, Chemosphere.

[56]  H. Blanck,et al.  Metagenomic sequencing of marine periphyton: taxonomic and functional insights into biofilm communities , 2015, Front. Microbiol..

[57]  G. Braus,et al.  One Juliet and four Romeos: VeA and its methyltransferases , 2015, Front. Microbiol..