The impact of primer design on amplicon-based metagenomic profiling accuracy: detailed insights into bifidobacterial community structure

Simple item page
dc.contributor.authorMancabelli, Leonardo
dc.contributor.authorMilani, Christian
dc.contributor.authorLugli, Gabriele A.
dc.contributor.authorFontana, Federico
dc.contributor.authorTurroni, Francesca
dc.contributor.authorvan Sinderen, Douwe
dc.contributor.authorVentura, Marco
dc.contributor.funderHorizon 2020
dc.contributor.funderScience Foundation Ireland
dc.date.accessioned2023-09-05T15:07:02Z
dc.date.available2023-09-05T15:39:13Zen
dc.date.available2023-09-05T15:07:02Z
dc.date.issued2020-01en
dc.date.updated2023-09-05T14:39:16Zen
dc.description.abstractNext Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.
dc.description.statusPeer revieweden
dc.description.versionPublished Version
dc.format.mimetypeapplication/pdfen
dc.identifier.articleid131
dc.identifier.citationMancabelli, L., Milani, C., Lugli, G.A., Fontana, F., Turroni, F., Van Sinderen, D. and Ventura, M. (2020) ‘The impact of primer design on amplicon-based metagenomic profiling accuracy: detailed insights into bifidobacterial community structure’, Microorganisms, 8(1), 13, (11pp). doi.: 10.3390/microorganisms8010131
dc.identifier.doi10.3390/microorganisms8010131en
dc.identifier.endpage11
dc.identifier.issued1
dc.identifier.journaltitleMicroorganisms
dc.identifier.startpage1
dc.identifier.urihttps://hdl.handle.net/10468/14926
dc.identifier.volume8
dc.language.isoenen
dc.publisherMDPI
dc.relation.projectinfo:eu-repo/grantAgreement/EC/H2020::CSA/696300/EU/The second coordination and support action for the JPI Healthy Diet for a Healthy Life/CSA JPI HDHL 2.0
dc.relation.projectinfo:eu-repo/grantAgreement/SFI/EU Joint Programming Initiative::A Healthy Diet for a Healthy Life JPI (HDHL-JPI)/15/JP-HDHL/3280/IE/Impact of early life on microbiome development and later health (EarlyMicroHealth)/
dc.relation.projectinfo:eu-repo/grantAgreement/SFI/SFI Research Centres/12/RC/2273/IE/Alimentary Pharmabiotic Centre (APC) - Interfacing Food & Medicine/
dc.rights© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subject16S rRNA profiling
dc.subjectBifidobacterium
dc.subjectMetagenomics
dc.subjectMicrobiota
dc.subjectPrimer pairs
dc.titleThe impact of primer design on amplicon-based metagenomic profiling accuracy: detailed insights into bifidobacterial community structure
dc.typeArticle (peer-reviewed)
Files
Original bundle
Now showing 1 - 2 of 2
Thumbnail Image
Name:
microorganisms-08-00131.pdf
Size:
11.32 MB
Format:
Adobe Portable Document Format
Description:
Published Version
Download
Thumbnail Image
Name:
microorganisms-08-00131-s001.zip
Size:
2.58 MB
Format:
http://www.iana.org/assignments/media-types/application/zip
Description:
Supplementary Materials
Download
Collections
APC Microbiome Ireland - Journal Articles
Microbiology - Journal Articles