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HAL Id: hal-02463373https://hal.archives-ouvertes.fr/hal-02463373
Submitted on 5 Jun 2020
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rpoB, a promising marker for analyzing the diversity ofbacterial 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 foranalyzing the diversity of bacterial communities by amplicon sequencing. 9. Colloque de l’AssociationFrancophone 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. Ogier1, S. Pagès1, M. Barret2 and S. Gaudriault1
1INRA, Université Montpellier, UMR1333 DGIMI, CC054, 34095 Montpellier Cedex 05, France2INRA, 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 rRNAgene is classically used to estimate bacterial diversity, but its low discriminating power forcertain bacterial genera and its variable copy number in prokaryotic genomes constituteimportant limitations (2). In this study, we assessed the potential benefit of a portion of therpoB gene as an alternative genetic marker. We first analyzed the sequence data generatedby metabarcoding with rpoB and 16S (V3V4 region) markers on an artificial bacterial DNAcomplex corresponding to 19 different phylogenetic taxa. We then compared theperformance of the rpoB and V3 V4 markers in an animal ecosystem model, the infectivejuveniles (IJs) of the entomopathogenic nematode Steinernema glaseri carrying thesymbiotic bacteria Xenorhabdus poinarii.
2. Analysis of Mock communitiesImpact 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 NematodesPreparation of 5 mock communities
Mix of genomic DNAs extractedfrom 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 rpoBmarker
7 ussp
76
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
234
Surface IJs cleaning (water)
* the numbers = OTU number
40% 80%
Illumina Miseq sequencing
Amplification of two genetic markers
16S v3v4 rpoB
Data analysis
Raw read processing Quality check
Chimera removal Read abundance filter (>0 005% of the whole data set)
DADA
FROG
Afaecalis
1607
0.050
Illumina_rpoBthreshold =0.1%sensitivity: 19/19Seq variants: 12Chimeras: 1
Xen
orhabdu
Acinetobactersp 460
0.050
Illumina_16Sthreshold =0.1%Sensitivity: 16/19Seq variants: 24Chimeras: 4
430 pb450 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 rpoBmarker, i.e. the 19 species which compose the mock are detected
OTU composition at the Phylum and Family levelBiological 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 soilsF
RE
E F
OR
M I
N S
OIL
S
x40
Xenorhabdus SteinernemaNon symbiotic bacteria(intercuticular space)
© S. Pagès
x400
0.75
0.5
0.25
AlcaligenaceaeBrucellaceaeCaulobacteraceaeComamodaceaePseudomonaceaeRhizobiaceaeSphingomodaceaeXanthomodaceaeYersiniaceaeEnterobacteriaceaeBurkholderiaceae
Relative ab
undan
ce(%
)
0.75
0.5
0.25
BacteroidetesProteobacteriaActinobacteriaFirmicutes
IJ infects insect hostIJs emerge fom insect cadaver
rpoB 16S rpoB 16S
Phylogenetic tree of the overall OTUsOTU redundancies(Sequences variants)
Delftia
815
Ochrobactrum
587
Similar bacterial compositions were obtained with both markers at the Phylum level (mainly Proteobacteria), but differences appear at the Family level
Bacteria released intoinsect hemolymph
Bacteria infection kills insect host
PA
RA
SIT
IC P
HA
SE
IN
INS
EC
TS
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 4 1 2 3 4 1 2 3 4
The OTU richness is dependent on the used marker (vary from 30 to 55 OTUs)
OTU richness
Enterobacteriaceae 206
phomonas155
Rhizobiaceae 53
0.050
O
0.10
rpoBmarker24 potential OTUs12 seq variants
16S marker23 potential OTUs46 seq variants
10
20
30
40
50
60
70
Number of OTU
s
The pipeline tools (FROGS vs DADA2) 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 isavailable 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. luminescens and X. bovieniiinstead of X. poinarii))
Stenotrop
Only rpoBmarker detects the bacterial symbiont X. poinarii
0 Probable over estimation of OTU richness with 16S marker