High throughput CNV analysis - 2 billion data
points in 20 weeks
John Anson PhD, Oxford Gene Technology
Who are OGT and what do we do?
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Founded in 1995 by Professor Ed Southern
Fundamental IP on microarray technology
High throughput microarray servicing
Cytogenetics (CytoSure™) products and service
Single cell analysis (CellScribe ™)
OGT Business Model
Clinical & Genomics solutions
Applications of arrays from the lab to the clinic
Biomarker discovery
Protein and DNA biomarkers
Digital microarrays/single cell analysis
for analysing genomic events at single cell level
Licensing fundamental array patents
OGT is a molecular
medicine company
providing advanced
clinical genetics
services and
developing innovative
molecular diagnostics
Many issues face scientists investigating the genetic
basis of disease
• Most diseases have a
complex molecular basis
• Large scale multi-faceted
studies are required
• Methods employed need to
be sensitive, specific,
reliable, with a high
capacity and quick
turnaround
Conventional biomarkers studied
DNA
• Single nucleotide polymorphisms (SNPs)
RNA
• Messenger RNA (mRNA) markers
Protein
Metabolites
Prospective biomarkers
DNA
• Single nucleotide polymorphisms (SNPs)
• Epigenetic (methylation)
• Copy number variation (CNV)
RNA
• Messenger RNA (mRNA) markers
• MicroRNA (miRNA) markers
Protein
Metabolites
Copy Number Variation
Genotyping CNVs using aCGH
Test genomic DNA
Reference genomic DNA
Label with Cy5
Label with Cy3
Hybridise
Wash and scan
Feature extract & load into analysis software
Challenge: Scaling up aCGH for high throughput CNV
genotyping
• Labelling protocol needs to be
“robot friendly”
• Automated slide washing required
• Tracking of samples and assay
performance essential
• Ozone monitoring and control
• Powerful computational capability
required for data for analysis and
data storage
High Throughput CNV Service Project
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24 leading human geneticists
Copy number variations (CNVs)
>20,000 DNA samples, and associated reference sample
7 disease areas
3,000 controls
• >1000 DNA samples processed per week
OGT in collaboration with Agilent technologies provided high throughput
servicing of the WTCCC CNV arrays
High throughput samples are prepared
using automation
• Experimental error is minimised,
promoting consistency
• Metrics fed directly into LIMS
assuring accuracy
• >600 samples and controls per
day
Quality control is paramount at every step
• QC check to ensure that dye
incorporation and yield metrics are
achieved prior to hybridisation
• Data consistency is continually
monitored, immediately highlighting
variation and minimising waste of:
• sample
• expense
• time.
Hybridisation capacity of ~300 slides in parallel
• Hybridisation oven temperatures
are monitored constantly to
ensure:
• optimal hybridisation
conditions
• more reliable results
Automated slide washing ensuring reduced variability
• Automated slide washing eliminates
manual handling and hence,
potential variation
• Provides batch to batch
reproducibility for large studies
• Ozone-controlled and monitored
environment preserving dyes and
data quality
• Wash buffer, reagent temperatures,
agitation rates and wash times are
controlled and reported in LIMS
Fully automated array scanning, data download & analysis
• Ozone-controlled
environment protects against
dye degradation
• Data securely stored and
backed up in real-time
(batches of 192 slides)
• Data transferred rapidly and
securely
LIMS provides full traceability at all stages
• Samples are barcoded and tracked throughout the process
• All sample and array QC metrics
• All mastermix and consumable batches used
• All equipment utilised
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PCR machine (and calibration)
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Hybridisation oven (continuous temperature recording)
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Centrifuges
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Spectrophotometers (and frequent calibration)
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Automated washing stations (temperatures, and speeds)
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Scanners
• Ozone levels (washing and scanning environments)
LIMS is customisable & provides full traceability at all stages
>40 QC checks
completed for every
sample processed –
complete reassurance
LIMS provides full traceability at every stage
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Customised modules for: plate handling, unique array IDs, hybridisation oven, and scanner
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QC on all reagents and consumables: digest and labelling mixes,
incubation block, clean-up kit, cot-1 solution and wash solutions
Example in-process control metrics
• OGT internal control sample pass rate = >98%
0.5
Heat
block
failure
DLRSD
0.4
Purification
module
failure
0.3
POOR
GOOD
0.2
EXCELLENT
0.1
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Wk1
Wk19
Time
DLRS = derivative log ratio spread
This metric is a measure of the reliability of the data to detect & call CNV aberrations
Signal Intensity (Cy3)
Signal Intensity of Control Samples
EXCELLENT
GOOD
POOR
Wk1
Wk19
Time
Signal intensity is consistently high and gives excellent QC metrics throughout 19 weeks.
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Signal-To-Noise (Cy3)
Signal to Noise Ratio of Control Samples
EXCELLENT
GOOD
POOR
Wk1
Wk19
Time
Signal-To-Noise is consistently excellent (based in Agilent QC metrics) throughout 19 weeks.
This ensures optimal dynamic range of data enabling greater confidence in CNV calling.
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Some numbers...
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20,146 samples processed
40,292 labelling reactions
20,146 arrays hybridised
2.1x109 data points generated
5.1Terabytes of data shipped
Processing time 20 weeks
WTCCC HapMap Study published October 2009
Findings from the study
• Any two genomes differ by more than 1000 CNVs, or around 0.8%
of a person's genome sequence.
• Most of these CNVs are deletions, with a minority being
duplications.
• Two consequences are particularly striking in this study of
apparently healthy people:
• 75 regions have jumped around in the genomes of these samples
• more than 250 genes can lose one of the two copies without obvious
consequences and a further 56 genes can fuse together potentially to
form new composite genes
But...
“We have not found large numbers of common CNVs that we can tie
strongly to disease. There remains much to be discovered and much
to understand and our freely available genotyped collection will drive
that discovery. “
Matt Hurles, Wellcome Trust Sanger Institute
Products from OGT Services
• Using OGT Services to identify growing
applications for Products
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Gene expression
Array Comparative Genome Hybridisation (aCGH)
ChIP on chip
Methylation/CpG islands
miRNA profiling
Karyotyping – gross changes
Example of a normal karyotype
Example of Down’s Syndrome (trisomy 21),
the most common numerical abnormality found
in newborns. It is characterised by an extra
chromosome 21.
Karyotyping - deletions
http://www.pathology.washington.edu/galleries/Cytogallery/main.ph
p?file=digeorge%20syndrome Accessed 19.09.06
CytoSure family
CytoSureTM Cytogenetics range
• Arrays – OGT designed, manufactured by Agilent:
• Syndrome Plus
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2x105k developed with Pr. Joris Vermeesch Belgium
4x44k ISCA approved
4x180k
4x180k ISCA approved
• Chromosome X
• 4x44k
• 2x105k
• Aneuploidy (new)
• 8x15k (220Kb)
• DMD (new)
• 4x44k developed with Emory
• Labelling kit & Ancillaries
• Analysis Software
• Oligome custom arrays
CytoSure software designed for cytogenetics
Syndrome
Genes
Recom hot
CNVs
Confirmation
Patient data
(del)
Patient data
(dup)
Data courtesy Greenwood clinic, South Carolina
Summary of OGT’s capability
Designed to meet the need of large scale CNV genotyping
studies:
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Data quality – rigorous QC critical (proven in WTCCC study)
Throughput – currently >1,200 samples per week (saleable)
Data handling and storage – proprietary LIMS
Custom array design service – from whole genome exploration to
focussed interrogation in key regions of interest
Where next for high throughput CNV genotyping?
• Several large scale projects on the horizon
• New array formats provide flexibility between content coverage and
cost
• CytoSure software adapted for data analysis
• Routine processing of clinical samples
Impact of next gen sequencing?
Potential for a symbiotic relationship
Acknowledgements
• High throughput CNV genotyping:
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Graham Speight
Nicole Sparkes
Andrew Rogers
Sandra Lam
Tom Nicholls
John Shovelton
• CytoSure
• Doug Hurd
• John Shovelton
• Volker Brenner
Thank you