Improving Species Level‐taxonomic Assignment from 16S rRNA Sequencing Technologies

Analysis of the bacterial community from a 16S rRNA gene sequencing technologies requires comparing the reads to a reference database. The challenging task involved in annotation relies on the currently available tools and 16S rRNA databases: SILVA, Greengenes and RDP. A successful annotation depend...

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Veröffentlicht in:Current protocols 2023-11, Vol.3 (11), p.e930-n/a
Hauptverfasser: Bars‐Cortina, David, Moratalla‐Navarro, Ferran, García‐Serrano, Ainhoa, Mach, Núria, Riobó‐Mayo, Lois, Vea‐Barbany, Jordi, Rius‐Sansalvador, Blanca, Murcia, Silvia, Obón‐Santacana, Mireia, Moreno, Victor
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container_issue 11
container_start_page e930
container_title Current protocols
container_volume 3
creator Bars‐Cortina, David
Moratalla‐Navarro, Ferran
García‐Serrano, Ainhoa
Mach, Núria
Riobó‐Mayo, Lois
Vea‐Barbany, Jordi
Rius‐Sansalvador, Blanca
Murcia, Silvia
Obón‐Santacana, Mireia
Moreno, Victor
description Analysis of the bacterial community from a 16S rRNA gene sequencing technologies requires comparing the reads to a reference database. The challenging task involved in annotation relies on the currently available tools and 16S rRNA databases: SILVA, Greengenes and RDP. A successful annotation depends on the quality of the database. For instance, Greengenes and RDP have not been updated since 2013 and 2016, respectively. In addition, the nature of 16S sequencing technologies (short reads) focuses mainly on the V3‐V4 hypervariable region sequencing and hinders the species assignment, in contrast to whole shotgun metagenome sequencing. Here, we combine the results of three standard protocols for 16S rRNA amplicon annotation that utilize homology‐based methods, and we propose a new re‐annotation strategy to enlarge the percentage of amplicon sequence variants (ASV) classified up to the species level. Following the pattern (reference) method: DADA2 pipeline and SILVA v.138.1 reference database classification (Basic Protocol 1), our method maps the ASV sequences to custom nucleotide BLAST with the SILVA v.138.1 (Basic Protocol 2), and to the 16S database of Bacteria and Archaea of NCBI RefSeq Targeted Loci Project databases (Basic Protocol 3). This new re‐annotation workflow was tested in 16S rRNA amplicon data from 156 human fecal samples. The proposed new strategy achieved an increase of nearly eight times the proportion of ASV classified at the species level in contrast to the reference method for the database used in the present research. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample inference and taxonomic profiling through DADA2 algorithm. Basic Protocol 2: Custom BLASTN database creation and ASV taxonomical assignment. Basic Protocol 3: ASV taxonomical assignment using NCBI RefSeq Targeted Loci Project database. Basic Protocol 4: Definitive selection of lineages among the three methods.
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subjects 16S rRNA gene amplicons
Biodiversity
Computer Science
DADA2
Humanities and Social Sciences
Life Sciences
Medicin och hälsovetenskap
metagenomics
Methods and statistics
Microbiology and Parasitology
microbiota composition
Quantitative Methods
species‐classification
Systematics, Phylogenetics and taxonomy
title Improving Species Level‐taxonomic Assignment from 16S rRNA Sequencing Technologies
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