New Lab Tech. Bruce L. Akey BS MS DVM Director, Texas A&M Veterinary Medical Diagnostic Laboratory

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Theodore Wilcox
6 years ago
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1 New Lab Tech Bruce L. Akey BS MS DVM Director, Texas A&M Veterinary Medical Diagnostic Laboratory WSLHA July 9, 2015

2 Usefulness Se/Sp Throughput Turnaround

3 New Technologies MALDI TOF Next Generation Sequencing (NGS) Luminex Microarray

4 Matrix Assisted Laser Desorption-Ionization Time Of Flight

5 MALDI-TOF

6 MALDI-TOF

7 MALDI-TOF

8 MALDI-TOF

9 MALDI-TOF Strengths Simplifies workflow Faster turnaround Cheaper/isolate ID More objective semiquantitative Expanding database Weaknesses Expensive initial investment Mixed cultures can be problematic Database still lacking in some genera/species ID only, still requires separate workup for antibiotic sensitivity Only as good as original culture

10 Next Generation Sequencing Genomics/Metagenomics

12 NGS Methods

13 NGS

14 NGS "Mapping Reads" by Suspencewl - Own work. Licensed under CC0 via Wikimedia Commons -

15 NGS - Ion Sequencing

0 via Wikimedia Commons - https://commons.wikimedia.

16 NGS - Ion Sequencing "DNTP incorporation hydrogen magnitude" by David Tack - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons - rporation_hydrogen_magnitude.svg

17 "AHL DNA" by George Church DNA.GIF NGS - Nanopore NanoporeSequencing.htm

18 Metagenomics Protocol for Metagenomic Virus Detection in Clinical Specimens Kohl C, Brinkmann A, Dabrowski PW, Radonić A, Nitsche A, Kurth A Emerg Infect Dis [Internet] Jan DOI: /eid

19 Opinion: Genomics in the Clinic Next-generation sequencing diagnostics are already being used, and patients are ready. The Scientist March 18, 2013 In the NGS world, everyone marvels at the dramatic fall in sequencing costs: it is 100 million times cheaper than it was only 10 years ago when the human genome project was just wrapping up. Much of that improvement in efficiency is realized in the sheer number of genes that can be measured at the same time. Two years ago, clinical labs relied on Sanger sequencing to sequence one or two genes, whereas today, NGS allows us to sequence the entire exome of roughly 23,000 genes for the same cost, giving the geneticist far more information to consider. Other costs associated with sequencing are not falling as quickly, however. If you truly account for every dollar the library preparation, target enrichment, sequencing machine depreciation, reagents, lab personnel, power, building costs, data transfer, computation, storage, backup, and time for clinical geneticists to analyze the data an exome quoted at $700 actually costs a total of $2,882. The software used to analyze NGS data in the clinic must meet higher standards than in research environments. No false negatives. Few false positives. Intuitive reports that physicians can understand.

20 NGS Strengths ID of unknowns Organism doesn t have to be viable/culturable Can target one or multiple organisms Strain differences Potential for both ID and sensitivity or other characteristics (toxins) Weaknesses Expensive initial setup Additional SME Low throughput/workflow Only as good as published databases of sequences Error rate Interpretation QA practices not yet robust Data volume/analysis $$$/sample

Clinic/Field Testing Portable DNA Sequencer Can ID Bacteria and Viruses A palm-sized, nanopore-based USB device can recognize E. coli, cowpox, and vaccinia.

Three poxviruses (cowpox, vaccinia-mva, and vaccinia-lister) were identified and differentiated down to the strain level, despite over 98% identity between the vaccinia strains.

21 Clinic/Field Testing Portable DNA Sequencer Can ID Bacteria and Viruses A palm-sized, nanopore-based USB device can recognize E. coli, cowpox, and vaccinia. The Scientist March 26, 2015 Using a 6 hour sequencing run time, sufficient data were generated to identify an E. coli sample down to the species level from 16S rdna amplicons. Three poxviruses (cowpox, vaccinia-mva, and vaccinia-lister) were identified and differentiated down to the strain level, despite over 98% identity between the vaccinia strains. The ability to differentiate strains by amplicon sequencing on the MinION was accomplished despite an observed per-base error rate of approximately 30%. GigaScience 2015, 4:12 doi: /s z

22 LUMINEX

23 LUMINEX - Multiplexing

24 LUMINEX

25 LUMINEX - MAGPIX

26 LUMINEX Strengths Flexibility multiplexing High Throughput Workflow integration $/sample Results/interpretation of known type Quantitative and qualitative Weaknesses Moderate startup costs Kit/reagent development needed

27 Microarray

28 Microarray - multiplexing

29 Microarray - multiplexing

Microarray - multiplexing OpenArray technology uses a microscope slide sized plate with 3,072 through-holes. Each plate contains 48 subarrays, each with 64 through-holes.

30 Microarray - multiplexing OpenArray technology uses a microscope slide sized plate with 3,072 through-holes. Each plate contains 48 subarrays, each with 64 through-holes. Each through-hole is 300 μm in diameter and 300 μm deep and is treated with hydrophilic and hydrophobic coatings. Reagents are retained in the through-holes via surface tension. One OpenArray plate can hold as many samples as can eight traditional 384-well plates.

31 Microarray - multiplexing

32 Microarray - multiplexing Multiplex screening for blood-borne viral, bacterial, and protozoan parasites using an OpenArray platform Grigorenko E, Fisher C, Patel S, Chancey C, Rios M, Nakhasi HL, Duncan RC J Mol Diagn Jan;16(1): DOI: /j.jmoldx Epub 2013 Oct 30

33 Microarray Strengths Flexibility multiplexing High Throughput $/sample Results/interpretation of known type Quantitative and qualitative Weaknesses Expensive startup costs Custom array development

34 Test Technology Luminex Microarray RNA seq RNA seq Luminex Microarray Luminex Microarray RNA seq MALDI-TOF Luminex Microarray RNA seq

35 Thank You

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