“cgmp of genomic microbial identification · whole genome sequencing –tracing • whole genome...

© PharmOut 2018

“cGMP" of Genomic Microbial Identification

Presented by Wayne McKenzie

& Tara Cassidy , August 2018

© 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

© 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




RECEPTION

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

© 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


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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”


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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


© 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.


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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


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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)


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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


MALDI-TOF – More suitable for less critical environments / screening

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MALDI-TOF Strain typing (P.acnes)

• Flex Analysis • Offline Software


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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


© 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


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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


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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


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Microbial Diversity – ATCC Mock Community Results

MGX deliver world class “best practice” Microbial Diversity


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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


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Gut Microbiome – Microbial DiversityBelieved to have huge impacts on health and wellbeing being implicated in many diseases and disorders.

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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

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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


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NGS – Challenges met by cGMP approach Sampling

DNA Extraction

Primer Selection

Sequencing Approach

Bioinformatics selection


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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


© 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


“cgmp of genomic microbial identification · whole genome sequencing –tracing • whole genome...

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