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rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing
Jean-Claude Ogier, Sylvie Pages, Matthieu Barret, Sophie Gaudriault
To cite this version:
Jean-Claude Ogier, Sylvie Pages, Matthieu Barret, Sophie Gaudriault. rpoB, a promising marker for
analyzing the diversity of bacterial communities by amplicon sequencing. 9. Colloque de l’Association
Francophone d’Ecologie Microbienne, Nov 2019, Bussang, France. �hal-02463373�
RPOB, A PROMISING MARKER FOR ANALYZING THE DIVERSITY OF BACTERIAL COMMUNITIES BY AMPLICON SEQUENCING (1)
J.C. Ogier
1, S. Pagès
1, M. Barret
2and S. Gaudriault
11
INRA, Université Montpellier, UMR1333 DGIMI, CC054, 34095 Montpellier Cedex 05, France
2
INRA, Agrocampus Ouest, Université d'Angers, UMR –IRHS, 49071 Beaucouzé, France
OBJECTIVES: Microbiome composition is frequently studied by the amplification and high‐throughput sequencing of specific molecular markers (metabarcoding). The 16S rRNA gene is classically used to estimate bacterial diversity, but its low discriminating power for certain bacterial genera and its variable copy number in prokaryotic genomes constitute important limitations (2). In this study, we assessed the potential benefit of a portion of the rpoB gene as an alternative genetic marker. We first analyzed the sequence data generated by metabarcoding with rpoB and 16S (V3V4 region) markers on an artificial bacterial DNA complex corresponding to 19 different phylogenetic taxa. We then compared the performance of the rpoB and V3 V4 markers in an animal ecosystem model, the infective juveniles (IJs) of the entomopathogenic nematode Steinernema glaseri carrying the symbiotic bacteria Xenorhabdus poinarii.
2. Analysis of Mock communities
Impact on OTU taxonomic affiliations and observed OTU richness
mock1 mock2 mock3 mock4 mock5 Expected rpoB
mock1 Expected 16S
rpoB
10 20 30 40 50 60
number of OTUs
Expected
symbiotic bacteria Xenorhabdus poinarii.
1. Experimental design for metabarcoding analysis
IJs crushing and Quick extract Kit DNA extraction from Nematodes Preparation of 5 mock communities
Mix of genomic DNAs extracted from 19 bacterial species 5 mocks differing in the proportions of the taxa
(5000 IJs)
OTU richness overestimated, especially for 16S marker
Taxonomic assignation level is better with rpoB marker
7 ussp76
Phylogenetic tree of the overall OTUs
mock2 mock3 mock4 mock5
20% 60%
> order Family Genus Species 16S
0
The optimal read abundance threshold to individual sample is 0.1%
mock1
mock5 2 3
4 Surface IJs cleaning (water)
*the numbers = OTU number
40% 80%
Illumina Miseq sequencing Amplification of two genetic markers
16S
v3v4rpoB
Data analysis
Raw read processing
Quality check
Chimera removal
Read abundance filter ( >0 005% of the whole data set) D
A D A
F R O G
Afaecalis1607
0.050
Illumina_rpoB
threshold =0.1%sensitivity: 19/19 Seq variants: 12
Chimeras: 1
Xenorhabdu
Acinetobactersp460 0.050
Illumina_16S
threshold =0.1%Sensitivity: 16/19 Seq variants: 24
Chimeras: 4
430 pb 450 pb
3 replicates per mock 4 replicates per nematode
3. Description of the nematodes IJ microbiota (Steinernema glaseri, 4 replicates)
Read abundance filter ( >0.005% of the whole data set)
2 S
Taxonomic assignment ( RDP Blast, 97% similarity) rpoB database*
(45 000 seq) Many additional OTUs are observed with V3V4 marker, corresponding to chimeras and variant sequences
Sensitivity is better with rpoB marker, i.e. the 19 species which compose the mock are detected
OTU composition at the Phylum and Family level Biological cycle of entomopathogenic nematodes
*created for this study and in open access at http://frogs.toulouse.inra.fr
Read abundance filter for individual sample (>0.1% and >1% ) SILVA database
rpoB 16S rpoB 16S
Infective juvenile (IJ) searching for insect prey in soils
FREE FORM IN SOILS x40
Xenorhabdus Steinernema
Non symbiotic bacteria (intercuticular space)
© S. Pagès
x400
0.75
0.5
0.25
Alcaligenaceae Brucellaceae Caulobacteraceae Comamodaceae Pseudomonaceae Rhizobiaceae Sphingomodaceae Xanthomodaceae Yersiniaceae Enterobacteriaceae Burkholderiaceae
Re la ti ve abunda nce (% )
0.75
0.5
0.25
Bacteroidetes Proteobacteria Actinobacteria Firmicutes
IJ infects insect host IJs emerge fom insect cadaver
rpoB 16S rpoB 16S
Phylogenetic tree of the overall OTUs
OTU redundancies (Sequences variants)
Delftia815
Ochrobactrum587
Similar bacterial compositions were obtained with both markers at the Phylum level (mainly Proteobacteria), but differences appear at the Family level
Bacteria released into insect hemolymph
Bacteria infection kills insect host
PARASITIC PHASE IN INSECTS
IJs emerge fom insect cadaver
IJ development and colonization
Nematode growth using insect and bacterial biomass as food
1 2 3 4
1 2 3 41 2 3 4
1 2 3 4The OTU richness is dependent on the used marker (vary from 30 to 55 OTUs)
OTU richness
Enterobacteriaceae206
phomonas155
Rhizobiaceae53 0.050
O
0.10
rpoB marker 24 potential OTUs
12 seq variants
16S marker 23 potential OTUs
46 seq variants
10 20 30 40 50 60 70
N umbe r of OT U s The pipeline tools (FROGS vsDADA2) does not influence the OTU richness
References: (1) Ogier et al., 2019. rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing. BMC Microbiol. 2019 Jul 29;19(1):171. doi: 10.1186/s12866-019-1546-z.
(2) Roux et al., 2011, Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems. FEMS Microbiol Ecology. 78. 617–628
CONCLUSIONS: The use of rpoB gene for metabarcoding analysis is a promising approach to accurately explore the diversity of bacterial communities because of its best discriminating power and the reduction of bias compared to the 16S marker. We created a database which includes 45,000 rpoB sequences covering the large diversity of available prokaryotic genomes. This database is available from the FROGS website (http://frogs.toulouse.inra.fr/).
Numerous sequence variants with the 16S marker (overestimation of the OTU diversity)
these sequence variants generate OTU identification errors (e.g. identification of P. luminescensand X. bovienii instead of X. poinarii))
Stenotrop
