Présentation le 4 juillet 2019Deciphering intra-species bacterial diversity using [i]gyrB[/i] amplicon sequencing. JOBIM 2019.  

Rué, Olivier

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Archive Ouverte en Sciences de l'Information et de la Communication

Deciphering intra-species bacterial diversity
using gyrB amplicon sequencing
A comparative analysis with 16S rDNA V3-V4 amplicon
sequencing
Olivier Rué
July 4, 2019
REDucing food LOSSES by microbial spoilage prediction
2 / 24
REDucing food LOSSES by microbial spoilage prediction
3 / 24
Diversity using metabarcoding
4 / 24
Knowledge
Predominance of Bacteria
Between 15 to 80 species
Limited phylogenetic diversity
mainly Firmicutes and
Proteobacteria
Large majority is known and
cultivable
16S rDNA pro ling limitations
Multiple copies of 16S
Amplification biases
Over-estimation of diversity
 Genus-level resolution
Meat spoilage
 Food quality is highly correlated to strain's diversity ! 
Need an alternative solution to assess with precision the
strain's diversity
5 / 24
Potential advantages of gyrB (gyrase β subunit)
Characteristics
Gene encoding for DNA gyrase (essential to DNA replication)
Ubiquist
Present in Bacteria, Eukaryota and sometimes Archae
280 nt long - less secondary structures (chimera)
One copy (rare exceptions)
No amplification of chloroplast and mitochondrial rDNA
Higher nucleotide mutation drift than 16S
6 / 24
Experienced in seed microbiota
Highly degenerated primers
(~2,000 times) designed in a
conserved AA region
Potential advantages of gyrB (gyrase β subunit)
gyrB metabarcoding ?
7 / 24
Proof of concept
Questions :
1. Reflect the real diversity ?
2. Quantification biases ?
3. Sub-species level reachable ?
8 / 24
4 Mock communities 15 food samples as 'unknown'
tests
Design of the study
One mixture with large strain's
diversity expected
9 / 24
Bioinformatics work ow
FROGS to obtain Operational Taxonomic Units
10 / 24
gyrB databank
No ready-to-use resources
gyrB public databanks not complete
Use of whole sequenced genomes
A lot of complete genomes in public repositories
gyrB well annotated
Other sequences to add
Unpublished genomes, home-made databanks...
Sequence, species and strain needed
NCBI taxonomy resources
Offer the ability to easily growth the content on demand 
11 / 24
gyrB databank
12 / 24
Results
13 / 24
Re ect the real diversity 
Number of observed OTUs (species richness) Diversity of genus-level taxa (Genus richness)
16S gyrB 16S gyrB
Mock1 (15 species) 59 42 15 15
Mock2 (15 species) 77 53 16 15
Mock3 (15 species) 56 37 15 14
Mock4 (5 species) 65 50 8 5
Mock5 (complex community) 110 229 33 40
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Re ect the real diversity 
Number of observed OTUs (species richness) Diversity of genus-level taxa (Genus richness)
16S gyrB 16S gyrB
Mock1 (15 species) 59 42 15 15
Mock2 (15 species) 77 53 16 15
Mock3 (15 species) 56 37 15 14
Mock4 (5 species) 65 50 8 5
Mock5 (complex community) 110 229 33 40
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Re ect the real diversity 
Number of observed OTUs (species richness) Diversity of genus-level taxa (Genus richness)
16S gyrB 16S gyrB
Mock1 (15 species) 59 42 15 15
Mock2 (15 species) 77 53 16 15
Mock3 (15 species) 56 37 15 14
Mock4 (5 species) 65 50 8 5
Mock5 (complex community) 110 229 33 40
16 / 24
Re ect the real diversity 
Number of observed OTUs (species richness) Diversity of genus-level taxa (Genus richness)
16S gyrB 16S gyrB
Mock1 (15 species) 59 42 15 15
Mock2 (15 species) 77 53 16 15
Mock3 (15 species) 56 37 15 14
Mock4 (5 species) 65 50 8 5
Mock5 (complex community) 110 229 33 40
17 / 24
Quanti cation biases 
quantitative PCR
18 / 24
Amplification of parE (gyrb
paralog)
No impact on quantification
Ampli cation biases 
19 / 24
Amplification of parE (gyrb
paralog)
No impact on quantification
Specific to Firmicutes
Ampli cation biases 
20 / 24
Sub-species level 
21 / 24
Sub-species level 
16S
gyrB
22 / 24
Conclusions / Take home messages
At the genus level : 16S (V3-V4) and gyrB performs identically 
Taxonomic assignment performed 100% at the species level with gyrB 
Tracking and tracing of strain’s diversity was possible 
Easy use of complete genomes for building a specific gyrB databank 
Guidelines
For not well known food or other microbiota: always include a 16S rDNA
control !
Validated for Firmicutes and Proteobacteria.
Needs validation for other phyla.
23 / 24
Thanks
Simon Poirier (Post-Doc MICALIS, publication author)
INRA units and teams
  
REDLOSSES consortium
JOBIM programme comitee
24 / 24


Deciphering intra-species bacterial diversity using gyrB amplicon sequencing

https://hal.archives-ouvertes.fr/hal-02266901/file/2019_Rue_JOBIM.pdf

Deciphering intra-species bacterial diversity using gyrB amplicon sequencing

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