Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
Slide 1 © PharmOut 2018
“cGMP" of Genomic Microbial Identification
Presented by Wayne McKenzie
& Tara Cassidy , August 2018
Slide 2 © PharmOut 2018
Microgenetix – Expertise in Microbial Genomics
Recognized Market Leader and expert in Microbial Genomics / IdentificationProven delivery in leading Pharma / Food / Medical Device / Consultant Laboratory market segmentsReference laboratory for major Melbourne Hospital and leading consultant laboratoriesStrong regulatory experience cGMP / HACCP Expert staff - Microbiologists & BiotechnologistscGMP Compliant Facilities & EquipmentMultiple Platform approach for Microbial IdentificationValidated and qualified systems4500 sequencing runs – 250,000 identifications in the last 10 yearsEarly technology adopter – first company in Australia to commercially sequence microorganisms
© Microgenetix 2018
Slide 3 © PharmOut 2018
Dedicated Microbial Identification Laboratory
GENOMICS GENERAL LABORATORY AREA
GENOMICS SEQUENCING ROOM
GENOMICS PCR ROOM
PC2 PATHOGEN REFERENCE
LABORATORY
OFFICE
Manometer
Return Air Grille
Ceiling HEPA Filter
Laboratory Bench
Incubator
U p
LABORATORY MANAGER
Incubator
Incubator
-80° C Freezer
STORE ROOM
Class II Safety Cabinet
Class II Safety Cabinet
Class II Safety Cabinet
Class II Safety Cabinet
RECEPTION
Class II Safety Cabinet Class II Safety
Cabinet
Class II Safety Cabinet
MALDI-TOF
Sample Receipt
Sample Preparation
Stag
ing
DN
A Extraction St
rain
Typ
ing R
eal-time PC
R
PCR
Pre
para
tion
3130xl Genetic Analyzer
3130xl Genetic Analyzer
Thermal Cycler
Thermal Cycler
Thermal Cycler
Thermal Cycler
-15 Pa > 0 Pa
0 Pa
cGMP Compliant
HEPA Filtered and segregated
work zones
Double barrier work zones with
dedicated equipment
Flush clean finish
Validated equipment and
procedures
Proven Capacity and Redundancy
Fully documented and
implemented Quality System
“Dedicated and Controlled environment”© Microgenetix 2018
Slide 4 © PharmOut 2018
Microbial Genomics at MGX
2007
85-90% speciation rate
1800 bacterial species Library
500bp 16S sequence
Limited identification platforms
Limited knowledge of microbial diversity
26 region sequencing for Fungi
4 day result turnaround
Limited client isolate history
2 MASM Microbiologist
2018
Over 250,000 isolates identified
Close to 100% speciation rate
›6000 MGX bacterial library
800bp 16S sequence
Multiple identification platforms
26S/ ITS region sequencing for Fungi
Next day result turnaround same day
Significant client isolate history
7 MASM Microbiologists
© Microgenetix 2018
Slide 5 © PharmOut 2018
Multiplatform Approach
Sanger Sequencing - (2x 3130xl Genetic Analysers)
MicroSEQ (10 years)
MGX Library
MGX 16S (5 years)
MALDI-TOF Proteolytic Identification
Species Identification
Phylotyping
Real Time PCR Methods (Specific Target)
Probiotics
Next Generation Sequencing
Validated DNA Extraction
16S Diversity profiling (NGS)
Whole Genome Sequencing
REP PCR – Strain Typing“State of art Microbial Identification”
© Microgenetix 2018
Slide 6 © PharmOut 2018
Purification of sequencing product
Purification of PCR product
Gold Standard 16S SequencingMicroSEQ® - Validated System Workflow
Starting material: sample from colony or pure culture
PCR with MicroSeq® PCR kit (1 reaction / sample)
Sequencing reaction with MicroSeq® sequencing kit
(2 reactions / sample: forward and reverse)
Capillary electrophoresis on Genetic Analyzer
DNA isolation with PrepMan™ Ultra kit
Library Search
Report (Hit List)
Data analysis in MicroSeq® ID software
© Microgenetix 2018
Slide 7 © PharmOut 2018
MGX 16S Sequencing Optimisation MicroSEQ delivers CFR part 11 compliance via Test Kit Reagents, Validated Equipment, Curated Library and Software. Following 10+ years and 100,000’s of identifications, MGX have optimised sequencing reflecting world best practice approaches including:
MGX Bacterial Library
Validated library - (6000+ entries with over 4000+ MGX custom entries) provides close to a 100% next day speciation result
History - MGX library accessions added from Australian isolates on an ongoing basis
Tracking - Great comparison tool - we keep every sequence!
High level of accuracy and reproducibility – Improved species confidence influencing 41% of MicroSEQspecies results eg.
© Microgenetix 2018
Slide 8 © PharmOut 2018
Optimisation of Sanger Sequencing
MGX 16S & ITS Generic Sequencing reagents
Cost Effective - Non CFR compliant but uses the same validated equipment, systems, staff and software.
MGX 16S 800 - Increased Accuracy via Longer Reads
MGX QC Sanger sequencing data shows reliable long read lengths are achievable (up to 900 base pairs)
A longer read inclusive of the MicroSEQ 500 (v1-v3) read achieves an additional 300-400 base pair reads targeting more (v1,v2,v3 & v4) 16S gene regions providing:
= Higher resolution = More informative than MicroSEQ sequence reads alone
Longer reads overcome polymorphisms & short consensus issues (frequent with some genera eg. Bacillus sp.)
Better exploits the powerful MGX 16S Full gene custom library
© Microgenetix 2018
Slide 9 © PharmOut 2018
MGX 16S 800 Validation Experience
MGX Validation Results
Highly accurate (100%)
Highly reproducible (100%)
Satisfies USP 1113 monograph requirement for verification of microbial identification methods
Accuracy - Equivalent to MicroSEQ: over 95% of isolates (n>50) in the validation study obtaining the same top match “best hit” or an improved identification basis (>13%)
Existing MicroSEQ User Acceptance Criteria is appropriate
Ongoing approach to library development
Recommendations
Result - high speciation outcome with next day result guarantee
Improved identification basis for many genera, eg. many Bacillus species, and is expected to provide improved identification basis for micro-organisms with polymorphisms in the 16s rDNA gene
Cost - Equivalent to “true” cost of MALDI
TAT - fast turn around (next day)
© Microgenetix 2018
Slide 10 © PharmOut 2018
MALDI-TOF Proteolytic Identification (3 years)
MALDI-TOF advantages seen for our clients:
Bruker MALDI-TOF is CFR Compliant (cGMP)
Rapid “Same Day” identification service
Low sample presentation cost with “Direct Method”
Phylotyping capability for some organisms
MALDI-TOF challenges:
Performance Issues - Colony age, Sample re-streaking time delays up to 5
days with fall through (Up to 15%) requiring sequencing
Sample Preparation / extraction methods - can improve reproducibility
Score Value – Poor consensus re acceptance criteria (OEM / IVD)
Poor demarcation / reproducibility with some genera
Lower speciation confidence
MSP Library Updates / Suitability – Necessary with relevance to client environs
Service Issues - laser tuning – result & costs impacts
© Microgenetix 2018
MALDI-TOF – More suitable for less critical environments / screening
Slide 11 © PharmOut 2018
MALDI-TOF Strain typing (P.acnes)
• Flex Analysis • Offline Software
© Microgenetix 2018
Slide 12 © PharmOut 2018
Nucleic Acid Based Techniques
Increasingly pharmacopoeia are providing for NAD / Genotypic Microbial Identification Methods
USP 113 describes methods to validate and verify nucleic acid test assays
DNA genotyping – Microbial Identification
Traditional PCR – Active Identification - confirmation of probiotic / vaccine strains
Real-Time PCR – targeted detection of biological contaminants e.g. Mycoplasma
© Microgenetix 2018
Slide 13 © PharmOut 2018
USP dietary supplement and FCC monographs for probiotic strains
USP published 8 monographs in 2015 in the Food Chemical Codex (FCC) which provides an active specificity test challenging the suitability of many probiotics
Lactobacillus acidophilus LA-14 & NCFM
Lactobacillus rhamnosus HN001
Lactobacillus paracasei Lpc-37
Bifidobacterium animalis ssp. lactis
(HN019, Bi-07, Bl-04)
Bacillus coagulans GBI-30,6086
Monographs contain (At strain level):
Description Identification AssaySpecific Tests Labelling requirement
© Microgenetix 2018
Slide 14 © PharmOut 2018
Nucleic Acid-Based Identification of Probiotic Strains
Identification Process
1. Targeted primer sets
2. PCR Amplification
3. DNA Electrophoresis
4. DNA Sequencing (SNP Analysis)
Acceptance Criteria for B.lactis HN019
• PCR Preparation for primer set 1 gives an amplification product of 351base pairs with a SNP identified as CTTCAGATTTTAGGC
• PCR Preparation for primer set 2 gives an amplification product of 531base pairs with a SNP identified as GCCCGCTCAAACGAA
Primer set 1 Primer set 2
© Microgenetix 2018
Slide 15 © PharmOut 2018
NGS – Next Generation Sequencing
Applications
Environmental monitoring (best practice)
Gut Microbiome (Humans & Animals)
Probiotic organism identification / evaluation
Fermented Foods & Beverages (Control / Drift)
Whole genome (Trace - Highly Specific)
Key Measurements
DAS – Microbial Diversity, Abundance and Significance
© Microgenetix 2018
Slide 16 © PharmOut 2018
Microbial Diversity – ATCC Mock Community Results
MGX deliver world class “best practice” Microbial Diversity
© Microgenetix 2018
Slide 17 © PharmOut 2018
NGS Microbial Diversity provides a “best practice” (Illumina - 2x300bp reads) approach to environmental monitoring - Surface Swab Results
Sample no.: 1 2 3 4 5 6 Sample no.: 1 2 3 4 5 6
16S Bacterial Diversity ex Swab ITS Fungal Diversity ex Swab
© Microgenetix 2018
Slide 18 © PharmOut 2018
Gut Microbiome – Microbial DiversityBelieved to have huge impacts on health and wellbeing being implicated in many diseases and disorders.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
Human Gut Phylum Level Comparison
Actinobacteria Bacteroidetes Chloroflexi
Cyanobacteria Euryarchaeota Firmicutes
Nitrospirae Proteobacteria Tenericutes
Unclassified at Phylum level Verrucomicrobia
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
Human Gut Genus Level Comparison
Alkaliphilus Bacteroides Bifidobacterium
Blautia Catenibacterium Collinsella
Coprococcus Faecalibacterium Methanobrevibacter
Oscillospira Phascolarctobacterium Prevotella
Ruminococcus Succinivibrio Unclassified at Genus level
© Microgenetix 2018
Slide 19 © PharmOut 2018
Probiotics / fermented foods
Microbial diverse samples - Can be accurately tested for the first time!
© Microgenetix 2018
Slide 20 © PharmOut 2018
NGS – Challenges met by cGMP approach Sampling
DNA Extraction
Primer Selection
Sequencing Approach
Bioinformatics selection
© Microgenetix 2018
Slide 21 © PharmOut 2018
NGS & Bionformatics – MGX Advanced Pipeline / Significant organisms
C.difficle ABUNDANCE (%) C.botulinum ABUNDANCE (%)
TEST SAMPLE MGX MSR/BaseSpace QIIME MGX MSR/BaseSpace QIIME
1 0.04 0.0 0.0 0.01 0.0 0.0
2 0.51 0.0 0.0 0.23 0.0 0.0
3 0.15 0.0 0.0 0.09 0.0 0.0
© Microgenetix 2018
Slide 22 © PharmOut 2018
Whole Genome Sequencing – Tracing
• Whole genome sequencing reveals the complete DNA make-up of an organism, enabling us to better understand variations both within and between species.
• Provides a high-resolution, base-by-base view of the genome
• Captures both large and small variants that might otherwise be missed
• Identifies potential causative variants for further follow-on studies of gene expression and regulation mechanisms
• Delivers large volumes of data in a short amount of time to support assembly of novel genomes
© Microgenetix 2018
Slide 23 © PharmOut 2018
Thankyou
Wayne McKenzie / Tara Cassidy
Microgenetix
www.microgenetix.net