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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