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Title: 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 » as these will increase the number of environments in which bacterial communities can be successfully studied.« less

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
. Mon . "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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Cited by: 29 works
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    Works referencing / citing this record:

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    Comparative analysis of DNA extraction methods to study the body surface microbiota of insects: A case study with ant cuticular bacteria
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    Social behaviour in bees influences the abundance of Sodalis (Enterobacteriaceae) symbionts
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    Community analysis of microbial sharing and specialization in a Costa Rican ant–plant–hemipteran symbiosis
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    Assessment of a 16S rRNA amplicon Illumina sequencing procedure for studying the microbiome of a symbiont-rich aphid genus
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    Comparative analysis of DNA extraction methods to study the body surface microbiota of insects: A case study with ant cuticular bacteria
    journal, June 2017


    Gut microbiota of dung beetles correspond to dietary specializations of adults and larvae
    journal, November 2016


    Dietary specialization in mutualistic acacia-ants affects relative abundance but not identity of host-associated bacteria
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    Establishing microbial composition measurement standards with reference frames
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