U.S. Department of EnergyOffice of Scientific and Technical Information
DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure
Abstract
The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications suchmore »
- Publication Date:
- Research Org.:
- Argonne National Lab. (ANL), Argonne, IL (United States)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1212882
- Grant/Contract Number:
- AC02-06CH11357; NSF1050243; NSF1311417
- Resource Type:
- Journal Article: Accepted Manuscript
- Journal Name:
- MicrobiologyOpen
- Additional Journal Information:
- Journal Volume: 3; Journal Issue: 6; Journal ID: ISSN 2045-8827
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 60 APPLIED LIFE SCIENCES; 16S rRNA; ants; DNA extraction; Earth Microbiome Project; host-associated bacteria; insects; microbiome
Citation Formats
. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure. United States: N. p., 2014.
Web. doi:10.1002/mbo3.216.
. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure. United States. https://doi.org/10.1002/mbo3.216
. 2014.
"DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure". United States. https://doi.org/10.1002/mbo3.216. https://www.osti.gov/servlets/purl/1212882.
@article{osti_1212882,
title = {DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure},
author = {},
abstractNote = {The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.},
doi = {10.1002/mbo3.216},
url = {https://www.osti.gov/biblio/1212882},
journal = {MicrobiologyOpen},
issn = {2045-8827},
number = 6,
volume = 3,
place = {United States},
year = {2014},
month = {12}
}
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Works referenced in this record:
Comparative Analysis of Human Gut Microbiota by Barcoded Pyrosequencing
journal, July 2008
- Andersson, Anders F.; Lindberg, Mathilda; Jakobsson, Hedvig
- PLoS ONE, Vol. 3, Issue 7
Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens
journal, March 2012
- Aylward, Frank O.; Burnum, Kristin E.; Scott, Jarrod J.
- The ISME Journal, Vol. 6, Issue 9
Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads
journal, April 2011
- Bartram, Andrea K.; Lynch, Michael D. J.; Stearns, Jennifer C.
- Applied and Environmental Microbiology, Vol. 77, Issue 11
PyNAST: a flexible tool for aligning sequences to a template alignment
journal, November 2009
- Caporaso, J. G.; Bittinger, K.; Bushman, F. D.
- Bioinformatics, Vol. 26, Issue 2
QIIME allows analysis of high-throughput community sequencing data
journal, April 2010
- Caporaso, J. Gregory; Kuczynski, Justin; Stombaugh, Jesse
- Nature Methods, Vol. 7, Issue 5
Moving pictures of the human microbiome
journal, January 2011
- Caporaso, J. Gregory; Lauber, Christian L.; Costello, Elizabeth K.
- Genome Biology, Vol. 12, Issue 5
Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms
journal, March 2012
- Caporaso, J. Gregory; Lauber, Christian L.; Walters, William A.
- The ISME Journal, Vol. 6, Issue 8
DNA extraction method affects microbial community profiles from soils and sediment
journal, October 2007
- Carrigg, Cora; Rice, Olivia; Kavanagh, Siobhán
- Applied Microbiology and Biotechnology, Vol. 77, Issue 4
Do diet and taxonomy influence insect gut bacterial communities?
journal, September 2012
- Colman, D. R.; Toolson, E. C.; Takacs-Vesbach, C. D.
- Molecular Ecology, Vol. 21, Issue 20
Bacterial Community Variation in Human Body Habitats Across Space and Time
journal, November 2009
- Costello, E. K.; Lauber, C. L.; Hamady, M.
- Science, Vol. 326, Issue 5960
Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB
journal, July 2006
- DeSantis, T. Z.; Hugenholtz, P.; Larsen, N.
- Applied and Environmental Microbiology, Vol. 72, Issue 7, p. 5069-5072
The gut microbiota of insects – diversity in structure and function
journal, September 2013
- Engel, Philipp; Moran, Nancy A.
- FEMS Microbiology Reviews, Vol. 37, Issue 5
Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil
journal, June 2009
- Feinstein, L. M.; Sul, W. J.; Blackwood, C. B.
- Applied and Environmental Microbiology, Vol. 75, Issue 16
Evidence for a persistent microbial seed bank throughout the global ocean
journal, March 2013
- Gibbons, S. M.; Caporaso, J. G.; Pirrung, M.
- Proceedings of the National Academy of Sciences, Vol. 110, Issue 12
The Earth Microbiome Project: Meeting report of the “1st EMP meeting on sample selection and acquisition” at Argonne National Laboratory October 6th 2010.
journal, January 2010
- Gilbert, Jack A.; Meyer, Folker; Jansson, Janet
- Standards in Genomic Sciences, Vol. 3, Issue 3
Meeting Report: The Terabase Metagenomics Workshop and the Vision of an Earth Microbiome Project
journal, January 2010
- Gilbert, Jack A.; Meyer, Folker; Antonopoulos, Dion
- Standards in Genomic Sciences, Vol. 3, Issue 3
The importance of metagenomic surveys to microbial ecology: or why Darwin would have been a metagenomic scientist
journal, June 2011
- Gilbert, Jack A.; O'Dor, Ronald; King, Nicholas
- Microbial Informatics and Experimentation, Vol. 1, Issue 1
Defining seasonal marine microbial community dynamics
journal, August 2011
- Gilbert, Jack A.; Steele, Joshua A.; Caporaso, J. Gregory
- The ISME Journal, Vol. 6, Issue 2
Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria
journal, August 2010
- Hazen, Terry C.; Dubinsky, Eric A.; DeSantis, Todd Z.
- Science, Vol. 330, Issue 6001
Correlates of gut community composition across an ant species ( Cephalotes varians ) elucidate causes and consequences of symbiotic variability
journal, December 2013
- Hu, Yi; Łukasik, Piotr; Moreau, Corrie S.
- Molecular Ecology, Vol. 23, Issue 6
The bacterial communities in plant phloem-sap-feeding insects
journal, January 2014
- Jing, Xiangfeng; Wong, Adam C-N; Chaston, John M.
- Molecular Ecology, Vol. 23, Issue 6
A Cross-Taxon Analysis of Insect-Associated Bacterial Diversity
journal, April 2013
- Jones, Ryan Thomas; Sanchez, Leticia Gonzales; Fierer, Noah
- PLoS ONE, Vol. 8, Issue 4
Surveying the Microbiome of Ants: Comparing 454 Pyrosequencing with Traditional Methods To Uncover Bacterial Diversity
journal, November 2012
- Kautz, Stefanie; Rubin, Benjamin E. R.; Russell, Jacob A.
- Applied and Environmental Microbiology, Vol. 79, Issue 2
Unlocking the potential of metagenomics through replicated experimental design
journal, June 2012
- Knight, Rob; Jansson, Janet; Field, Dawn
- Nature Biotechnology, Vol. 30, Issue 6
Supervised classification of human microbiota
journal, March 2011
- Knights, Dan; Costello, Elizabeth K.; Knight, Rob
- FEMS Microbiology Reviews, Vol. 35, Issue 2
Socially transmitted gut microbiota protect bumble bees against an intestinal parasite
journal, November 2011
- Koch, H.; Schmid-Hempel, P.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 48
Ecological effects on gut bacterial communities in wild bumblebee colonies
journal, June 2012
- Koch, Hauke; Cisarovsky, Gabriel; Schmid-Hempel, Paul
- Journal of Animal Ecology, Vol. 81, Issue 6
Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees
journal, January 2013
- Koch, Hauke; Abrol, Dharam P.; Li, Jilian
- Molecular Ecology, Vol. 22, Issue 7
Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform
journal, June 2013
- Kozich, James J.; Westcott, Sarah L.; Baxter, Nielson T.
- Applied and Environmental Microbiology, Vol. 79, Issue 17
Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples: Influence of short-term storage conditions on microbiota
journal, March 2010
- Lauber, Christian L.; Zhou, Nicholas; Gordon, Jeffrey I.
- FEMS Microbiology Letters, Vol. 307, Issue 1
Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis
journal, October 2004
- de Lipthay, Julia R.; Enzinger, Christiane; Johnsen, Kaare
- Soil Biology and Biochemistry, Vol. 36, Issue 10
Short pyrosequencing reads suffice for accurate microbial community analysis
journal, August 2007
- Liu, Z.; Lozupone, C.; Hamady, M.
- Nucleic Acids Research, Vol. 35, Issue 18
The Crematogaster (Hymenoptera, Formicidae, Myrmicinae) of Costa Rica
journal, March 2003
- Longino, John T.
- Zootaxa, Vol. 151, Issue 1
Negative Effects of Sample Pooling on PCR-Based Estimates of Soil Microbial Richness and Community Structure
journal, February 2010
- Manter, D. K.; Weir, T. L.; Vivanco, J. M.
- Applied and Environmental Microbiology, Vol. 76, Issue 7
Establishment of Characteristic Gut Bacteria during Development of the Honeybee Worker
journal, February 2012
- Martinson, Vincent G.; Moy, Jamie; Moran, Nancy A.
- Applied and Environmental Microbiology, Vol. 78, Issue 8
DNA preservation: a test of commonly used preservatives for insects
journal, January 2013
- Moreau, Corrie S.; Wray, Brian D.; Czekanski-Moir, Jesse E.
- Invertebrate Systematics, Vol. 27, Issue 1
Diet Drives Convergence in Gut Microbiome Functions Across Mammalian Phylogeny and Within Humans
journal, May 2011
- Muegge, B. D.; Kuczynski, J.; Knights, D.
- Science, Vol. 332, Issue 6032
Investigating the Impact of Storage Conditions on Microbial Community Composition in Soil Samples
journal, July 2013
- Rubin, Benjamin E. R.; Gibbons, Sean M.; Kennedy, Suzanne
- PLoS ONE, Vol. 8, Issue 7
Bacterial gut symbionts are tightly linked with the evolution of herbivory in ants
journal, November 2009
- Russell, Jacob A.; Moreau, Corrie S.; Goldman-Huertas, Benjamin
- Proceedings of the National Academy of Sciences, Vol. 106, Issue 50
Stability and phylogenetic correlation in gut microbiota: lessons from ants and apes
journal, January 2014
- Sanders, Jon G.; Powell, Scott; Kronauer, Daniel J. C.
- Molecular Ecology, Vol. 23, Issue 6
Genomic variation landscape of the human gut microbiome
journal, December 2012
- Schloissnig, Siegfried; Arumugam, Manimozhiyan; Sunagawa, Shinichi
- Nature, Vol. 493, Issue 7430
Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities
journal, October 2009
- Schloss, P. D.; Westcott, S. L.; Ryabin, T.
- Applied and Environmental Microbiology, Vol. 75, Issue 23, p. 7537-7541
Reducing the Effects of PCR Amplification and Sequencing Artifacts on 16S rRNA-Based Studies
journal, December 2011
- Schloss, Patrick D.; Gevers, Dirk; Westcott, Sarah L.
- PLoS ONE, Vol. 6, Issue 12
An Insect Herbivore Microbiome with High Plant Biomass-Degrading Capacity
journal, September 2010
- Suen, Garret; Scott, Jarrod J.; Aylward, Frank O.
- PLoS Genetics, Vol. 6, Issue 9
The NIH Human Microbiome Project
journal, October 2009
- Peterson, J.; Garges, S.; Giovanni, M.
- Genome Research, Vol. 19, Issue 12
The Human Microbiome Project
journal, October 2007
- Turnbaugh, Peter J.; Ley, Ruth E.; Hamady, Micah
- Nature, Vol. 449, Issue 7164
Comparison of DNA Extraction Methods for Microbial Community Profiling with an Application to Pediatric Bronchoalveolar Lavage Samples
journal, April 2012
- Willner, Dana; Daly, Joshua; Whiley, David
- PLoS ONE, Vol. 7, Issue 4
Linking Long-Term Dietary Patterns with Gut Microbial Enterotypes
journal, September 2011
- Wu, G. D.; Chen, J.; Hoffmann, C.
- Science, Vol. 334, Issue 6052
Microbial Dysbiosis During Simian Immunodeficiency Virus Infection is Partially Reverted with Combination Anti-retroviral Therapy
journal, April 2020
- Blum, Faith C.; Hardy, Britney L.; Bishop-Lilly, Kimberly A.
- Scientific Reports, Vol. 10, Issue 1
Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads
journal, July 2011
- Bartram, Andrea K.; Lynch, Michael D. J.; Stearns, Jennifer C.
- Applied and Environmental Microbiology, Vol. 77, Issue 15
Works referencing / citing this record:
Influence of Host Plant on Thaumetopoea pityocampa Gut Bacterial Community
journal, July 2017
- Strano, Cinzia P.; Malacrinò, Antonino; Campolo, Orlando
- Microbial Ecology, Vol. 75, Issue 2
Comparison of two bacterial DNA extraction methods from non-polluted and polluted soils
journal, July 2017
- Mazziotti, Mélanie; Henry, Sonia; Laval-Gilly, Philippe
- Folia Microbiologica, Vol. 63, Issue 1
Establishing microbial composition measurement standards with reference frames
journal, June 2019
- Morton, James T.; Marotz, Clarisse; Washburne, Alex
- Nature Communications, Vol. 10, Issue 1
Diversity and Persistence of the Gut Microbiome of the Giant Neotropical Bullet Ant
journal, June 2017
- Moreau, Corrie S.; Rubin, Benjamin E. R.
- Integrative and Comparative Biology, Vol. 57, Issue 4
Dramatic Differences in Gut Bacterial Densities Correlate with Diet and Habitat in Rainforest Ants
journal, July 2017
- Sanders, Jon G.; Łukasik, Piotr; Frederickson, Megan E.
- Integrative and Comparative Biology, Vol. 57, Issue 4
Social behaviour in bees influences the abundance of Sodalis (Enterobacteriaceae) symbionts
journal, July 2018
- Rubin, Benjamin E. R.; Sanders, Jon G.; Turner, Kyle M.
- Royal Society Open Science, Vol. 5, Issue 7
Community analysis of microbial sharing and specialization in a Costa Rican ant–plant–hemipteran symbiosis
journal, March 2017
- Pringle, Elizabeth G.; Moreau, Corrie S.
- Proceedings of the Royal Society B: Biological Sciences, Vol. 284, Issue 1850
The role of tissue-specific microbiota in initial establishment success of Pacific oysters: Microbiota and oyster establishment
journal, January 2016
- Lokmer, Ana; Kuenzel, Sven; Baines, John F.
- Environmental Microbiology, Vol. 18, Issue 3
Assessment of a 16S rRNA amplicon Illumina sequencing procedure for studying the microbiome of a symbiont-rich aphid genus
journal, November 2015
- Jousselin, E.; Clamens, A. -L.; Galan, M.
- Molecular Ecology Resources, Vol. 16, Issue 3
Comparative analysis of DNA extraction methods to study the body surface microbiota of insects: A case study with ant cuticular bacteria
journal, June 2017
- Birer, Caroline; Tysklind, Niklas; Zinger, Lucie
- Molecular Ecology Resources, Vol. 17, Issue 6
Optimal extraction methods for the simultaneous analysis of DNA from diverse organisms and sample types
journal, February 2018
- Hermans, Syrie M.; Buckley, Hannah L.; Lear, Gavin
- Molecular Ecology Resources, Vol. 18, Issue 3
A common garden experiment with Porphyra umbilicalis (Rhodophyta) evaluates methods to study spatial differences in the macroalgal microbiome
journal, August 2018
- Quigley, Charlotte T. C.; Morrison, Hilary G.; Mendonça, Inara R.
- Journal of Phycology, Vol. 54, Issue 5
Gut microbiota of dung beetles correspond to dietary specializations of adults and larvae
journal, November 2016
- Shukla, Shantanu P.; Sanders, Jon G.; Byrne, Marcus J.
- Molecular Ecology, Vol. 25, Issue 24
Dietary specialization in mutualistic acacia-ants affects relative abundance but not identity of host-associated bacteria
journal, August 2018
- Rubin, Benjamin E. R.; Kautz, Stefanie; Wray, Brian D.
- Molecular Ecology, Vol. 28, Issue 4
Effects of Organic Amendments on Microbiota Associated with the Culex nigripalpus Mosquito Vector of the Saint Louis Encephalitis and West Nile Viruses
journal, February 2017
- Duguma, Dagne; Hall, Michael W.; Smartt, Chelsea T.
- mSphere, Vol. 2, Issue 1
Reptile-associated Borrelia species in the goanna tick (Bothriocroton undatum) from Sydney, Australia
journal, December 2017
- Panetta, Jessica L.; Šíma, Radek; Calvani, Nichola E. D.
- Parasites & Vectors, Vol. 10, Issue 1
Denitrification potential of the eastern oyster microbiome using a 16S rRNA gene based metabolic inference approach
journal, September 2017
- Arfken, Ann; Song, Bongkeun; Bowman, Jeff S.
- PLOS ONE, Vol. 12, Issue 9
Abundance and Diversity of Hydrogenotrophic Microorganisms in the Infant Gut before the Weaning Period Assessed by Denaturing Gradient Gel Electrophoresis and Quantitative PCR
journal, June 2017
- Sagheddu, Valeria; Patrone, Vania; Miragoli, Francesco
- Frontiers in Nutrition, Vol. 4
Characteristics of Microbial Communities of Pachygrontha antennata (Hemiptera: Pachygronthidae) in Relation to Habitat Variables
journal, November 2019
- Kang, Jae-Yeon; Kwon, Yong-Su; Jeong, Gilsang
- International Journal of Environmental Research and Public Health, Vol. 16, Issue 23
High-Resolution Profiling of Gut Bacterial Communities in an Invasive Beetle using PacBio SMRT Sequencing System
journal, August 2019
- Xu, Letian; Sun, Liuwei; Zhang, Shihan
- Insects, Vol. 10, Issue 8
Evidence-based recommendations on storing and handling specimens for analyses of insect microbiota
journal, January 2015
- Hammer, Tobin J.; Dickerson, Jacob C.; Fierer, Noah
- PeerJ, Vol. 3
Fungal community assembly in soils and roots under plant invasion and nitrogen deposition
journal, August 2019
- Phillips, Michala L.; Weber, Sӧren E.; Andrews, Lela V.
- Fungal Ecology, Vol. 40