1. Probiotics R&D:
from genome sequences to regulatory issues
BACKGROUND
 Technologies for characterization and identification at strain level
are fundamental for probiotics development.
 Genome sequencing is widely used since many service providers
offer it at affordable prizes.
 Whole genome sequence analysis allows the widest range of data
retrieval.
 Regulatoryauthoritiessuggestorrequiregenomesequenceanalysis.
Here some examples:
OPEN INNOVATION IN MOLECULAR MICROBIOLOGY
A. Del Casale1
, E. Salvetti1,2
, G. E. Felis 1,2
, S. Torriani 1,2
, F. Fracchetti 1
1 - Microbion SRL; 2 – Verona University Department of Biotechnology
microbion.it
PROBLEMs
 Many sequencing platforms with specific pros/cons, therefore
selection is very difficult.
 Sequencing providers usually don’t have microbiology skills for
analysis of probiotics relevant genes.
 Uncompleted, erroneous or ambiguous genome assembly leads to
wrong conclusions and can be resource-wasters.
 Nonspecificgenomicdatabasesarepopulatedwithwrongtaxonomic
data, leading to ineffective for genomic assessment.
SOLUTIONs
 No “one-fit-all”, experimental design and customisation are the key
elements for industrial-wise genome sequencing application.
 Combination of different sequencing platforms leads to the most
accurate results.
 Genome assembly needs to be validated by independent methods
such as Whole Genome Mapping.
 Using dedicated databases and only controlled reference genomes
speed-up the workflow.
CONCLUSION
Robust genome sequencing project allows:
 Regulatory compliant genomic analysis;
 Strain traceability, in supply-chain and clinical trials;
 Patents by unique and recognisable characters.
MORE INFO:
Microbion SRL – Verona University Spin-off
Strada Le Grazie 15, Verona, Italy
Tel.: (+39) 045 802 7930 - info@microbion.it
and visit us at Vitafoods 2015, 5-7 May - Geneva
OPEN INNOVATION IN MOLECULAR MICROBIOLOGY
Profiling genomic DNA restriction fragment (PFGE, ribotyping)
Profiling PCR-amplified DNA (RAPD, MLVA, ERIC-, REP-PCR)
Selective PCR-amplification of restriction fragments (AFLP)
Restriction analysis of DNA fragments (PCR-RFLP)
DNA Multi-Locus Sequence Typing (MLST)
House-keeping gene Multi-Locus Sequence Analysis (MLSA)
Amplified Ribosomal DNA Restriction Analysis (ARDRA)
rRNA gene sequencing analysis (SSU, LSU)
Genomic DNA-DNA Hybridisation (DDH)
Genomic DNA mol% G+C
Whole genome sequencing analysis
FDA 2012 – Guidance for industry: Early clinical trials with live biothe-
rapeutic products;
EFSA 2012 –Guidance on assessment of bacteria susceptibility to anti-
microbials of human and veterinary importance;
EFSA 2012 – Guidance on the safety assessment of Enterococcus fae-
cium in animal nutrition;
EFSA 2014 – Guidance on the assessment of the toxigenic potential of
Bacillus species used in animal nutrition.
Fam
ily
G
enius
SpeciesStrain
Sequencing A Sequencing B
Long reads
Low fidelity
Short reads
High fidelity
De-novo Hybrid assembly
Validation with
Whole Genome Mapping
Annotation
Regulatory compliant
gene analysis