Microgenetix Meaningful Microbial Diversity Gut Health for Good Health

Transcription

1 Microgenetix Meaningful Microbial Diversity Gut Health for Good Health J G (Greg) Taylor B.Sc., Grad.Dip.Bus.Admin., AAIFST, MASM. Tara Cassidy B.Sc (Animal Science), B.Sc (Hons), MASM. Sept 2018

2 Microgenetix Expertise in Microbial Identification Founded in 2007 (Greg Taylor / Wayne McKenzie) career microbiologists looking for best practice solutions in Microbial Identification Recognized Market Leader and expert in Microbial Genomics / Identification Validated & cgmp Compliant Facilities & Equipment Multiple Platform approach to Microbial Identification Expert & Competent staff Consultants with significant Industry Expertise / 5x MASM Microbiologists & Biotechnologists

3 p U Dedicated Microbial Identification Laboratory MALDI- TOF Class II Safety Cabinet Class II Safety Cabinet Class II Safety Cabinet cgmp Compliant Class II Safety Cabinet DNA Extraction Strain Typing Real-time PCR PCR Preparation Staging Class II Safety Cabinet Sample Preparation Sample Receipt Incubator Incubator GENOMICS GENERAL LABORATORY AREA HEPA Filtered and segregated work zones PC2 PATHOGEN REFERENCE LABORATORY 0 Pa > 0 Pa -15 Pa Incubator LABORATORY MANAGER OFFICE GENOMICS SEQUENCING ROOM 3130xl Genetic Analyzer GENOMICS PCR ROOM Thermal Cycler Thermal Cycler Thermal Cycler Double barrier work zones with dedicated equipment -80 C Freezer Class II Safety Cabinet 3130xl Genetic Analyzer Class II Safety Cabinet Thermal Cycler Flush clean finish STORE ROOM RECEPTION Manometer Return Air Grille Ceiling HEPA Filter Validated equipment and procedures Laboratory Bench Proven Capacity and Redundancy Fully documented and implemented Quality System Dedicated and Controlled environment

4 Multiplatform Approach PCR & Sanger Sequencing (2x 3130 Genetic Analysers) MALDI-TOF Technology Real Time PCR (Specific Target) REP PCR Strain Typing Validated DNA Extraction 16S / ITS Diversity profiling (NGS) Whole Genome Sequencing State of art Microbial Identification

5 ACCUGENIX MICROBIAL IDENTIFICATION & STRAIN TYPING Maximizing environmental control with unparalleled accuracy, strain typing for the most challenging organisms and sophisticated data management. AccuGENX-ID Genotypic ID AccuPRO-ID Proteotypic ID Axcess System Insourcing Solution AccuGENX-ST Strain Typing SLST or MLST Tracking and Trending Data Environmental Monitoring Management Tools 5 EVERY STEP OF THE WAY

6 Microbial Diversity Why?

7 HOW? Traditional Sequencing Identification of individual colonies Colony 1- K.sedentarius Colony 2- S.hominis Colony 3-S.epidermidis Etc. etc. Next Generation Sequencing Diversity profiling of microbial communities extracted straight from the matrix.

8 Probiotics / Fermented foods

9 Metagenomics has limitations The Journey towards overcoming limitations to provide a Valid and Meaningful NGS Service Validate Sampling & DNA Extraction Validate Sequencing Approach Understand Report Options Validate Bioinformatics Validate Primers

10 DNA Extraction

11 Primer Selection

12 Features / Differences Bioinformatics Pipeline considerations Analysis Pipeline MSR (MGX) BaseSpace (MGX) QIIME MGX Powered Analysis Pipeline Pipeline Classification Method Assignment-first Assignment-first Clustering-first Assignment-first Reference library / size Greengenes (6,000+ species) Greengenes (6,000+ species) Greengenes (6,000+ species) Cost Low Medium Medium High Analysis speed Slow (about 14 hrs) Slow (about 14 hrs) Slow (depends on processor speed of comp) NCBI with curation (15,000+ species) Fast (about 30 min) Reporting Cost effective Good but costlier Poor and costlier Detailed and expensive Accuracy Average Average Average High Reporting presentation Top 8 classified All classified matches Approx. Top 30 All classified matches Graphical presentation (individual) Pie Chart Interactive sunburst plot and bar chart classification Bar Chart Comparisons Hierarchical dendrogram Hierarchical dendrogram Bar Chart Bar Chart Reporting Options Limited Medium Limited Multiple Typical Results Doughnut Chart and interactive sunburst plot

13 Bioinformatics Pipeline considerations Pipeline classification method

14 Bioinformatics Pipeline Impacts Phylum Trial Sample 1 Trial Sample 2 QIIME MGX Advanced MSR/BaseSpace QIIME MGX Advanced MSR/BaseSpa ce Actinobacteria Bacteroidetes Firmicutes Proteobacteria Verrucomicrobia Tenericutes Cyanobacteria Lentisphaerae Nitrospirae

15 Bioinformatics - Pipeline considerations Species Heavily depending on library size and specificity Consider : number of species classified Number of species presented (cut-offs) Results classification

16 MGX - DAS (Diversity, Abundance, Significance) Key Measures (DAS) - 3 key microbial measures relevant to study of microbiomes: 1. Microbial Diversity 2. Microbial Abundance 3. The likely significance of the microorganism.

17 Bioinformatics Analysis Comparison MGX Greengenes Top Species Classification Results MGX Advanced Top Species Classification Results

18 Bioinformatics Analysis Significance TEST SAMPLE C.difficle ABUNDANCE (%) C.botulinum ABUNDANCE (%) MGX MSR/BaseSpace QIIME MGX MSR/BaseSpace QIIME TEST SAMPLE E.coli (%) Salmonella (%) MGX Greengenes MGX Advanced MGX Greengenes MGX Advanced 1 Not identified 5.78 Not identified Not identified 2 Not identified 2.76 Not identified Not identified

19 Applications - Probiotics / fermented foods Can be tested for the first time! Proof they contain / do what they claim!

20 Applications - Gut Microbiome Believed to have huge impacts on health and wellbeing being implicated in many diseases and disorders. Human Gut Phylum Level Comparison Human Gut Genus Level Comparison 100% 100% 90% 90% 80% 80% 70% 70% 60% 50% 40% 30% 20% 10% 0% Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 60% 50% 40% 30% 20% 10% 0% Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Actinobacteria Bacteroidetes Chloroflexi Cyanobacteria Euryarchaeota Firmicutes Nitrospirae Proteobacteria Tenericutes Unclassified at Phylum level Verrucomicrobia Alkaliphilus Bacteroides Bifidobacterium Blautia Catenibacterium Collinsella Coprococcus Faecalibacterium Methanobrevibacter Oscillospira Phascolarctobacterium Prevotella Ruminococcus Succinivibrio Unclassified at Genus level

21 Applications - effects of diet on gut biome

22 Applications Environmental Monitoring best practice environmental monitoring 16S Bacterial Diversity ex Swab ITS Fungal Diversity ex Swab

23 Fit For Purpose To give a meaningful diversity profiling result the lab needs to do what is required not only what they can do!

24 NGS Combating Challenges It all comes back to our cgmp approach Using a Strict cgmp Approach to these Limitations can be controlled

25 Thankyou. Questions?