Evaluation of 96‐well high‐throughput DNA extraction methods for 16S rRNA gene metabarcoding
Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis.
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Subjects: | U60 - Sciences de la vie et de la Terre, PCR, adn, séquence nucléotidique, Bacteria, formation des prix, http://aims.fao.org/aos/agrovoc/c_34079, http://aims.fao.org/aos/agrovoc/c_2347, http://aims.fao.org/aos/agrovoc/c_27583, http://aims.fao.org/aos/agrovoc/c_765, http://aims.fao.org/aos/agrovoc/c_6177, |
Online Access: | http://agritrop.cirad.fr/604803/ http://agritrop.cirad.fr/604803/7/604803.pdf |
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dig-cirad-fr-6048032024-01-29T05:57:38Z http://agritrop.cirad.fr/604803/ http://agritrop.cirad.fr/604803/ Evaluation of 96‐well high‐throughput DNA extraction methods for 16S rRNA gene metabarcoding. Chapuis Marie-Pierre, Benoit Laure, Galan Maxime. 2023. Molecular Ecology Resources, 23 (7) : 1509-1525.https://doi.org/10.1111/1755-0998.13812 <https://doi.org/10.1111/1755-0998.13812> Evaluation of 96‐well high‐throughput DNA extraction methods for 16S rRNA gene metabarcoding Chapuis, Marie-Pierre Benoit, Laure Galan, Maxime eng 2023 Molecular Ecology Resources U60 - Sciences de la vie et de la Terre PCR adn séquence nucléotidique Bacteria formation des prix http://aims.fao.org/aos/agrovoc/c_34079 http://aims.fao.org/aos/agrovoc/c_2347 http://aims.fao.org/aos/agrovoc/c_27583 http://aims.fao.org/aos/agrovoc/c_765 http://aims.fao.org/aos/agrovoc/c_6177 Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis. article info:eu-repo/semantics/article Journal Article info:eu-repo/semantics/publishedVersion http://agritrop.cirad.fr/604803/7/604803.pdf text cc_by_nc info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc/4.0/ https://doi.org/10.1111/1755-0998.13812 10.1111/1755-0998.13812 info:eu-repo/semantics/altIdentifier/doi/10.1111/1755-0998.13812 info:eu-repo/semantics/altIdentifier/purl/https://doi.org/10.1111/1755-0998.13812 info:eu-repo/semantics/reference/purl/https://doi.org/10.18167/DVN1/D31UAV info:eu-repo/grantAgreement///ANR-10-LABX-0004//(FRA) Mediterranean Center for Environment and Biodiversity/CeMEB |
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U60 - Sciences de la vie et de la Terre PCR adn séquence nucléotidique Bacteria formation des prix http://aims.fao.org/aos/agrovoc/c_34079 http://aims.fao.org/aos/agrovoc/c_2347 http://aims.fao.org/aos/agrovoc/c_27583 http://aims.fao.org/aos/agrovoc/c_765 http://aims.fao.org/aos/agrovoc/c_6177 U60 - Sciences de la vie et de la Terre PCR adn séquence nucléotidique Bacteria formation des prix http://aims.fao.org/aos/agrovoc/c_34079 http://aims.fao.org/aos/agrovoc/c_2347 http://aims.fao.org/aos/agrovoc/c_27583 http://aims.fao.org/aos/agrovoc/c_765 http://aims.fao.org/aos/agrovoc/c_6177 |
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U60 - Sciences de la vie et de la Terre PCR adn séquence nucléotidique Bacteria formation des prix http://aims.fao.org/aos/agrovoc/c_34079 http://aims.fao.org/aos/agrovoc/c_2347 http://aims.fao.org/aos/agrovoc/c_27583 http://aims.fao.org/aos/agrovoc/c_765 http://aims.fao.org/aos/agrovoc/c_6177 U60 - Sciences de la vie et de la Terre PCR adn séquence nucléotidique Bacteria formation des prix http://aims.fao.org/aos/agrovoc/c_34079 http://aims.fao.org/aos/agrovoc/c_2347 http://aims.fao.org/aos/agrovoc/c_27583 http://aims.fao.org/aos/agrovoc/c_765 http://aims.fao.org/aos/agrovoc/c_6177 Chapuis, Marie-Pierre Benoit, Laure Galan, Maxime Evaluation of 96‐well high‐throughput DNA extraction methods for 16S rRNA gene metabarcoding |
description |
Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis. |
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article |
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U60 - Sciences de la vie et de la Terre PCR adn séquence nucléotidique Bacteria formation des prix http://aims.fao.org/aos/agrovoc/c_34079 http://aims.fao.org/aos/agrovoc/c_2347 http://aims.fao.org/aos/agrovoc/c_27583 http://aims.fao.org/aos/agrovoc/c_765 http://aims.fao.org/aos/agrovoc/c_6177 |
author |
Chapuis, Marie-Pierre Benoit, Laure Galan, Maxime |
author_facet |
Chapuis, Marie-Pierre Benoit, Laure Galan, Maxime |
author_sort |
Chapuis, Marie-Pierre |
title |
Evaluation of 96‐well high‐throughput DNA extraction methods for 16S rRNA gene metabarcoding |
title_short |
Evaluation of 96‐well high‐throughput DNA extraction methods for 16S rRNA gene metabarcoding |
title_full |
Evaluation of 96‐well high‐throughput DNA extraction methods for 16S rRNA gene metabarcoding |
title_fullStr |
Evaluation of 96‐well high‐throughput DNA extraction methods for 16S rRNA gene metabarcoding |
title_full_unstemmed |
Evaluation of 96‐well high‐throughput DNA extraction methods for 16S rRNA gene metabarcoding |
title_sort |
evaluation of 96‐well high‐throughput dna extraction methods for 16s rrna gene metabarcoding |
url |
http://agritrop.cirad.fr/604803/ http://agritrop.cirad.fr/604803/7/604803.pdf |
work_keys_str_mv |
AT chapuismariepierre evaluationof96wellhighthroughputdnaextractionmethodsfor16srrnagenemetabarcoding AT benoitlaure evaluationof96wellhighthroughputdnaextractionmethodsfor16srrnagenemetabarcoding AT galanmaxime evaluationof96wellhighthroughputdnaextractionmethodsfor16srrnagenemetabarcoding |
_version_ |
1792500545764720640 |