from my presentation at the 4th Annual Diagnostics & Medical Devices Asia 2009 meeting in Singapore

1. Automation:
a key role for Molecular
Diagnostic Evolution
Patrick Merel, PhD
University Hospital of Bordeaux, France
Biomedical Innovation Platform (PTIB)
pmerel@mac.com
1
1

2. Molecular Diagnostic Projections
From IBC Life Sciences’
Projections for 2012
Discovery2Diagnostics conference,
Oct. 08
2007 micro arrays market 2012 micro arrays market
$660 millions (9%)
$160 millions (3%)
2007 MDx market 2012 MDx market
$3 billions $7 billions
2
credits: Harry Glorikian, managing partner of Scientia Advisors
2

3. MDx Growth Factors 3
Demand for molecular diagnostics and gene detection products is mainly determined by the
volume of tests performed in clinical labs
3

4. MDx Growth Factors 3
Demand for molecular diagnostics and gene detection products is mainly determined by the
volume of tests performed in clinical labs
The need for automated and easy-to-handle techniques
Optimized sample preparation, analysis, and data evaluation
Techniques that would diagnose disease condition and medical disorders quickly for
quick therapy decisions
Availability of molecular diagnostic tests for monitoring the therapeutic efficacy of
expensive drugs
New diagnostics tests, primarily in the infectious disease application area
Nanobiotechnology and biochips are also expected to drive future growth
Genomics and proteomics are the major drivers of the molecular diagnostic market
3

5. MDx Growth Factors 4
A major step in MDx evolution: Realtime PCR technologies
4

6. MDx Growth Factors 4
A major step in MDx evolution: Realtime PCR technologies
Simplification of the PCR process
no more post-PCR procedures, ie. gel electrophoresis or hybridization
Single instrument based, ie. a realtime PCR thermocycler
Combination of a realtime PCR thermocycler and a Nucleic Acid (NA) extraction
instrument for a potential full solution in MDx
rapid development of automated NA extraction platforms
slow progression of automated realtime PCR instruments
new area of interest, fully automated platforms including NA extraction and rtPCR
4

7. NA extraction automation
evolution 5
5

8. NA Extraction Procedure Evolution
6
From single sample process to microtiterplate
by centrifugation
to generic robotic workstation
using vacuum manifolds
to magnetic beads based procedures
Biomérieux’s Boom patent and PSS
Magtration technology being the major drivers
6

9. Dedicated NA extraction platform
evolution
7
Interstingly, this progress in NA
extraction automation did’nt come
from the diagnostic industry and has
firstly addressed, the research and
academic market.
Thus, even usefull, often poorly
adapted to routine diagnostic
requirements
7

10. Dedicated NA extraction platform
evolution
7
Interstingly, this progress in NA
extraction automation did’nt come
from the diagnostic industry and has
firstly addressed, the research and
academic market.
Thus, even usefull, often poorly
adapted to routine diagnostic
requirements
7

11. Dedicated NA extraction
platform evolution
8
The Diagnostic Industry have
started to address routine MDx
demands (high throughput), FDA
and CE-IVD labeling more recently.
With the early presence of Gen-
Probe TIGRIS in blood transfusion
setups.
New players are now pushing
f o r w a rd t h e r u l e s w i t h f u l l y
integrated platforms.
8

12. Dedicated NA extraction
platform evolution
8
The Diagnostic Industry have
started to address routine MDx
demands (high throughput), FDA
and CE-IVD labeling more recently.
With the early presence of Gen-
Probe TIGRIS in blood transfusion
setups.
New players are now pushing
f o r w a rd t h e r u l e s w i t h f u l l y
integrated platforms.
8

13. Dedicated NA extraction
platform evolution
8
The Diagnostic Industry have
started to address routine MDx
demands (high throughput), FDA
and CE-IVD labeling more recently.
With the early presence of Gen-
Probe TIGRIS in blood transfusion
setups.
New players are now pushing
f o r w a rd t h e r u l e s w i t h f u l l y
integrated platforms.
8

14. Realtime PCR
instrumentation
9
9

15. Realtime PCR
instrumentation
9
9

16. Realtime PCR
instrumentation
9
9

17. Molecular Diagnostic Instrument
Challenges 10
While it is difficult nowdays to run MDX procedures without an automated platform, some
challenges remain to be fully addressed:
- from blood to buccal cells, sample type and volume are numerous. Hard to find a
single and unique solution
- no clear standard for platform capacity, sample batch size, batch vs continuous
flow, consolidation on core facilities vs point of care ..etc
- realtime PCR supremacy from infectious diseases to genetic testing but difficult to
fully automate
- sequencing and micro-arrays based diagnostic still high cost and time demanding
- SNP detection required in many potential personalized medicine assays still looking
for the ultimate gold standard for routine testing
10

18. From 8 samples to 96 samples
various platforms
ABI very 1st
automated NA from Tecan, Beckman
extractor Coulter, Hamilton to
Qiagen
while Roche back to 24 batch
Magnapure 32 with Biomérieux
sample size Nuclisens?
being of a major which has proved to
impact in labs be popular
to continuous flow and continuous
for 1 to 72 flow from 1 to 96
samples samples
with Qiagen
with Roche Cobas QiaSymphony,
AmpliPrep QiaEnsemble?
11
11

19. G
Continuous flow for NA
extraction procedures
Gen-Probe
the answer for multiple sample batch sizes
a progress toward random access instruments?
Roche Diagnostic
12
continous
flow
continous
innovation
Qiagen
12

20. G
Continuous flow for NA
extraction procedures
Gen-Probe
the answer for multiple sample batch sizes
a progress toward random access instruments?
Roche Diagnostic
12
continous
flow
continous
innovation
Qiagen 2009
2008 2010?
12

21. What’s next with NA
extraction automation
No more extraction steps?
microfluidics technologies potential
No more PCR?
Fully integrated instrumentation
Integration with Clinical Chemistry
Automation?
13
13

22. Cepheid
GeneXpert & Infinity
a precursor in microfluidics and fully automated process for MDx in a
single instrument
From 1 sample to 16
on the GeneXpert
to random access
on the Infinity
Cepheid
14
14

23. Cepheid
GeneXpert & Infinity
a precursor in microfluidics and fully automated process for MDx in a
single instrument
From 1 sample to 16
on the GeneXpert
to random access
on the Infinity
Cepheid
14
14

24. Cepheid
GeneXpert & Infinity
a precursor in microfluidics and fully automated process for MDx in a
single instrument
From 1 sample to 16
on the GeneXpert
to random access
on the Infinity
Cepheid
14
14

25. Microfluidics in MDx 15
HandyLab Jaguar, integrated DNA
extraction and realtime PCR
process on Microchips
Iquum Liat Analyzer and Liat Flow
Cycler, a new concept of Lab-in-a-
tube for realtime PCR based assays
Nanosphere Verigene
System for FDA approved
nanotechnology based
MDX assays.
protein assays compatible technology
Direct hybridization, no
PCR steps required
15

26. New Technology for new
process in MDx
Revolutionizing molecular
analysis
16
A microfluidic cartridge for multiplexed clinical assays
Superior
Universal
Fluidic Multiplex
Advantages
Specimen
Manipulation Molecular
Urine
Analysis No PCR
Delivery Saliva
Minutes to result
Separation Whole blood
Genetic assay
Low LOD(fM, pg/ml)
Mixing Serum, Plasma
Immunoassay
Dynamic range
Concentration Culture media
Electrolyte, Ions
Electrokinetic Concentrator
Target binding
Sample brief and rapid sample
delivery
prep. Almost no DNA
extraction step
BioElectronics with realtime
flow impedance monitoring
<10 min total assay time
16

27. New Technology for new
process in MDx
Revolutionizing molecular
analysis
16
A microfluidic cartridge for multiplexed clinical assays
Superior
Universal
Fluidic Multiplex
Advantages
Specimen
Manipulation Molecular
Urine
Analysis No PCR
Delivery Saliva
Minutes to result
Separation Whole blood
Genetic assay
Low LOD(fM, pg/ml)
Mixing Serum, Plasma
Immunoassay
Dynamic range
Concentration Culture media
Electrolyte, Ions
Electrokinetic Concentrator
Target binding
Sample brief and rapid sample
delivery
prep. Almost no DNA
extraction step
BioElectronics with realtime
flow impedance monitoring
<10 min total assay time
16

28. Revolutionizing molecular
MDx-Automation-Connectivity
analysis
17
New concept of MDx and telecommunication
Device Level Data Transmission System Level
Wireless
communication
Physical cable connection
Telecommunication
Portable device Transmission Clinical data station
 Security protection1  Security protection1
 SSL encryption3
 User authentication2  User authentication2
 Integrity control5
 Data encryption3  Data encryption6
 Digital certificates2
 Authetication4  Audit control7
1-7: Security Standards for the Protection of Electronic Protected Health Information
17

29. From Clinical Chemistry Environment
to MDx 18
Introducing clinical chemistry instrumentation parts in MDx platform
The Vidiera NSP
Primary tubes on racks
NA extraction by filtration
Qiagen chemistry
Quantification by spectrometry
Normalization
PCR setup
Archieving
Beckman Coulter
18

30. Connectivity in High
Throughput MDx settings 19
Abbott Molecular
Siemens
19

31. Connectivity in High
Throughput MDx settings 20
Roche Diagnostic
20

32. Connectivity in High
Throughput MDx settings 20
Roche Diagnostic
20

33. MDx new fields of application 21
Personalized medicine has brought attention onto SNP analysis
procedures in MDx
Oncology developments have made gene expression analysis a
growing field for MDx
Emerging infectious diseases, drug resistance monitoring,
NextGeneration Sequencing instruments have renewed the
interest for sequence based MDx
21

34. MDx outside realtime PCR 22
SNP and Gene Expression assays are missing consensus
technology in routine diagnostic, except for low complexity
assays that are making use of realtime PCR.
Sequencing technologies mature, but processing is long
and missing automation.
The promise of NextGen sequencing technologies.
22

35. SNP analysis MicroArrays Sequencing
Monoplex to Low Multiplexing Affymetrix-Roche CYP450 Capillary sequencing driven
Realtime PCR very 1st FDA available kit still technical demanding
not fully automated
Custom Multiplex Flexibility Low-Cost solutions (manual)
ABI-SNPlex Greiner BioOne CE-IVD chips Core facility oriented task
Sequenom-iPlex-MassArray Eppendorf BioChip
Beckman Coulter-SNPStream LGLife Sciences Whole Genome Sequencing-
DrChip NextGeneration Sequencing
Low to None Flexibility but very technologies
high Multiplexing Low-Cost solutions (semi-automated) lot of interest
iLLumina-Golden Gate ClonDiag-Genomica CE-IVD chips future of modern medicine, personalized
iLLumina-Infinium Autogenomics IVD, RUO medicine
Affymetrix-GeneChip Osmetech IVD
Hybribio Still work to do on:
BioCore cost/patient
process automation
Newest alternatives (fully automated) BioIT process
Nanosphere CE-IVD
GeneFluidics RUO
23
23

36. Increasing interest for automated
micro-array processing 24
Osmetech
Autogenomics
Greiner BioOne ClonDiag Genomica Nanosphere
24

37. Roche GS-FLXti Roche GS-FLXti:
0.4 Gb/run Setup time: 3-4 d
1m reads @ 400b 0.4Gb/run
Run time: 10 hrs
images: 27 GB
$7768/run 0.4Gb Primary Analysis: 15 GB
$19.41/Mb PA CPU time: 220 hrs
$648k/inst. Final file size: 4 GB
AB Solid 3.0 AB Solid 3.0:
10-20 Gb/run Setup time: 3-5 d
NextGen 100m reads @ 50b 5-12.5 Gb/run/slide
Run time: 3.5-10 d
Sequencing images: 2.5 TB
$6873/run 5+5Gb Primary Analysis: 750 GB
$0,69/Mb PA CPU time: in run time
$599k/inst. Final file size: 140 GB
Illumina GA2:
Illumina GA2
5-10 Gb/run Setup time: 2-3 d
the future 60m reads @ 50b 6-11 Gb/run
Run time: 3-6 d
of MDx? images: 900 GB
$8250/run 5Gb
Primary Analysis: 350 GB
$0,33/Mb
PA CPU time: 100 hrs
$460k/inst. Final File Size: 75 GB
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25

38. Roche GS-FLXti Roche GS-FLXti:
0.4 Gb/run Setup time: 3-4 d
1m reads @ 400b 0.4Gb/run
Run time: 10 hrs
images: 27 GB
$7768/run 0.4Gb Primary Analysis: 15 GB
$19.41/Mb PA CPU time: 220 hrs
$648k/inst. Final file size: 4 GB
AB Solid 3.0 AB Solid 3.0:
10-20 Gb/run Setup time: 3-5 d
NextGen 100m reads @ 50b 5-12.5 Gb/run/slide
Run time: 3.5-10 d
Sequencing images: 2.5 TB
$6873/run 5+5Gb Primary Analysis: 750 GB
$0,69/Mb PA CPU time: in run time
$599k/inst. Final file size: 140 GB
Illumina GA2:
Illumina GA2
5-10 Gb/run Setup time: 2-3 d
the future 60m reads @ 50b 6-11 Gb/run
Run time: 3-6 d
of MDx? images: 900 GB
$8250/run 5Gb
Primary Analysis: 350 GB
$0,33/Mb
PA CPU time: 100 hrs
$460k/inst. Final File Size: 75 GB
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39. Numerous fields of applications
26

40. HIV Ultra Deep Sequencing
for drug resistance monitoring
4000X coverage
minority populations detection
27

41. NextGen Roche GS-FLX:
Sequencers
Workflow
Workflow 3-4 days (setup) + 1 day (run)
+IT steps:
1. Generation of a single-stranded template DNA library (~8-16 hours)
2. Emulsion-based clonal amplification of the library (~8 hours)
3. Data generation via sequencing-by-synthesis (9 hours)
4. Image and Base calling analysis (~8 hours)
GS-FLX Software
5. Data analysis using different bioinformatics tools
▪GS Reference Mapper
•Long Single Reads / Standard Shotgun (required input = 3–5μg,5μg recommended)
~1,000,000 single reads with an average read length of 400 bases
▪GS De Novo Assembler
•Paired End Reads (required input = 5μg @25 ng/μl or above, in TE; >10kb)
▪GS Amplicon Variant
◦3K Long-Tag Paired End Reads. Sequence 100 bases from each end of a 3,000 base span
on a single sequence read (Figure). Co-assemble GS FLX Titanium shotgun reads with 3K
Analyzer
Long-Tag Paired Ends reads from Standard series runs.
•Sequence Capture (required input = 3–5μg)
◦Roche NimbleGen Sequence Capture using a single microarray hybridization-based
enrichment process.
Third Party Software
•Amplicon Sequencing (1-5ng or 10-50ng)
◦The DNA-sample preparation for Amplicon Sequencing with the GS FLX System consists of a
simple PCR amplification reaction with special Fusion Primers. The Fusion Primer consists of a
20-25 bp target-specific sequence (3' end) and a 19 bp fixed sequence (Primer A or Primer B
on the 5' end).
28

42. NextGen Roche GS-FLX:
Sequencers
Workflow
Workflow 3-4 days (setup) + 1 day (run)
+IT steps:
1. Generation of a single-stranded template DNA library (~8-16 hours)
2. Emulsion-based clonal amplification of the library (~8 hours)
3. Data generation via sequencing-by-synthesis (9 hours)
4. Image and Base calling analysis (~8 hours)
GS-FLX Software
5. Data analysis using different bioinformatics tools
▪GS Reference Mapper
•Long Single Reads / Standard Shotgun (required input = 3–5μg,5μg recommended)
~1,000,000 single reads with an average read length of 400 bases
▪GS De Novo Assembler
•Paired End Reads (required input = 5μg @25 ng/μl or above, in TE; >10kb)
▪GS Amplicon Variant
◦3K Long-Tag Paired End Reads. Sequence 100 bases from each end of a 3,000 base span
on a single sequence read (Figure). Co-assemble GS FLX Titanium shotgun reads with 3K
Analyzer
Long-Tag Paired Ends reads from Standard series runs.
•Sequence Capture (required input = 3–5μg)
◦Roche NimbleGen Sequence Capture using a single microarray hybridization-based
enrichment process.
Third Party Software
•Amplicon Sequencing (1-5ng or 10-50ng)
◦The DNA-sample preparation for Amplicon Sequencing with the GS FLX System consists of a
simple PCR amplification reaction with special Fusion Primers. The Fusion Primer consists of a
20-25 bp target-specific sequence (3' end) and a 19 bp fixed sequence (Primer A or Primer B
on the 5' end).
28

43. Conclusion 29
Realtime PCR has been a revolution for MDx
New procedures and lab organization
A 2 step process: NA extraction and PCR/detection
Bringing a new capacity for simple automation
Huge development and options for NA extraction
mag. beads procedures as a standard
Integration of realtime PCR step through full automated instrument
29

44. Conclusion 30
Development of fully integrated instrument for MDx is bringing new concept
and technologies
Microfluidics, bioelectronics, nanotechnologies
Telecommunication integration vs clinical chemistry settings integration
The needed evolution of SNP procedures
Finally low cost micro-array platforms with options for semi and full
automation
The next upcoming revolution in MDx: making use of NextGeneration
instrumentation in routine clinical diagnostic settings
30