This document discusses clinical and consumer applications of microarrays and genotyping technologies. It provides an overview of genotyping and different technologies like PCR microarrays and SNP microarrays. It describes how microarrays are still useful despite the rise of sequencing due to their low cost, high throughput, and ability to test millions of markers. The document outlines several applications of microarrays like direct-to-consumer testing, pharmacogenetics, and clinical sequencing. It also discusses challenges and trends in these areas like global initiatives to increase genomic data sharing.
3. Alexander Axt
• 8 years working in microarrays as a lab manager and business
development associate
• Processed over 500k samples on microarrays
• Clinically validated the first cytogenetics test on a microarray
• Works with over 2 dozen direct to consumer companies on
their microarray testing, both for clinical and non-clinical
applications
4. Genotyping
Genotyping is the process of
determining which genetic variants
an individual possesses.
Most common for of genotyping is
single nucleotide polymorphism
(SNP) genotyping which is a single
base pair mutation at a specific
locus, usually consisting of two
alleles.
Currently over 335 million SNPs
identified in the human genome.
Source: https://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi
5. Anecdotal Genotyping vs Sequencing Growth
0
5000
10000
15000
20000
25000
2004 2006 2008 2010 2012 2014 2016
Genotyping
Sequencing
Year
Number of
Citations
in PubMed
6. Cost of Microarrays vs. Sequencing
2005: Microarrays > $1,500 per sample
2018: Microarrays < $50 per sample
Microarrays are now highly cost
effective for large-scale studies
Whole Genome Sequencing ~ $10 million per sample
Whole Genome Sequencing < $1,000 per sample
WGS is still not cost effective
to test everyday consumers
7. Microarrays in a Sequencing World
•Mature Technology
•Low Cost
•Covers millions of markers in a single assay
•High Accuracy
•High Reproducibility
•High Throughput
•Smaller Data Sets
•Easier Analysis
•Used for a number of applications including: SNP
genotyping, copy number variations, gene expression,
microbiome, and methylation
9. Technologies – PCR
TaqMan
•DNA strand is denatured
•Primer is attached
•Taq Polymerase replicates strand
•Replication releases a fluorophore
that is detected.
•Probe principle relies on the 5´–3´
exonuclease activity of Taq
polymerase to cleave a dual-
labeled probe during hybridization
to the complementary target
sequence and fluorophore-based
detection
10. Technologies – PCR
Real Time – qPCR
Multiplexing Options
◦ Fluidigm EP1
◦ 24 - 96 markers
◦ Multiplexed by integrated fluidic circuits
(IFCs)
◦ Thermo Fisher Open Array
◦ 16-256 markers
◦ Multiplexed in a single well
11. Technologies –
Microarrays
The basic principles of the DNA
microarray. These are the convergence
of DNA hybridization, fluorescence
microscopy, and solid surface DNA
capture. The three mandatory
components of the SNP arrays are
1. An array containing immobilized allele-
specific oligonucleotide (ASO) probes.
2. Fragmented nucleic acid sequences of
target, labelled with fluorescent dyes.
3. A detection system that records and
interprets the hybridization signal.
12. Technologies – Microarrays
•Probes are fixed to a solid surface
•1k – 5 million unique probe markers
•Costs between $20-$300 per sample
depending on application
•Applications: SNP genotyping, gene
expression, methylation, CNV, miRNA,
pharmacogenetics, targeted panels, GWAS.
•Over 200 over the shelf products between the
two leading manufactures Illumina and
Affymetrix.
14. Bead Preparation and Array Production
• Unique oligo for each bead type
• Bead Pool can be > 1,000,000 bead types
• Random self-assembly of beads
• Average 15 to 30 beads of each type
• Functional validation of array
ProbeAddress
Target
15. Photolithography
UV light is passed through a lithographic mask that
acts as a filter to either transmit or block the light from
the chemically protected microarray surface (wafer).
The sequential application of specific lithographic
masks determines the order of sequence synthesis on
the wafer surface.
During the chemical synthesis cycle, UV light removes
the protecting groups (squares) from the array surface,
allowing the addition of a single protected nucleotide
as it is washed over the microarray.
Sequential rounds of light deprotection, changes in the
filtering patterns of the masks, and single nucleotide
additions form microarray features with specific 20-
25bp probes.
20. Hybridization
• Sample is hybridized to a 30-50 mer probe tiled on the array
• Complimentary genetic sequence binds to its’ probe set
• 9 mer solution probe pairs a specific “hapten” to a specific base
• Each probe set has 2-15 replicated probes
32. Direct to Consumer (DTC) Genetics Market Size
GLOBALLY GLOBALLY
Blue Ocean
Opportunity
$2 -7 Billion $1.5 Billion
Consumers
80M new users per year
Spending $60 per year
Pharma and Research
Partnerships
Personalized Consumer
Goods Partnerships
Source Genomics 2.0 UBS Report 2016
33. Specimens:
Wide range of sample type options
High per sample success rates and call rates across sample types
Note: performance of FFPE samples vary widely, very difficult to predict ahead of
time
Blood Swab
Saliva FFPE
Sample Types
34. Direct to Consumer Array
Content
◦ Contains ‘backbone’ from the Precision Medicine Research
Array
◦ Supplemented by client specific SNPs
◦ Proprietary content
◦ >900K SNPs
Model
◦ Laboratory runs same array for each client (genetics consumer
companies) and parses out specific SNP data to respective
clients (genetics consumer companies) who then report out
data to actual consumers
◦ Economies of scale
◦ End to end solution beginning with saliva kit or swab
35. Clinical Applications:
Dementia Array
Identify individuals at risk of cognitive decline,
Alzheimer’s disease and other dementias
>130,000 SNPs
Utilization of genotyping array on Axiom GeneTitan
platform
36. Pharmacogenetics – Quick Background
Precision pharmacogenetics (PGx)
genotyping enables a clinician to provide
a patient with optimized treatment by
maximizing drug efficacy and
minimizing adverse events
Over 300 actionable genetic variants
with dosing guidelines on FDA-approved
medications
18%
82%
Affected by actionable
pharmacogenes
Not affected by
actionable
pharmacogenes
7%
93%
FDA-approved medications (n=1,200) Prescriptions in the United States (n=4 billion)
Relling & Evans, Nature, 2015
37. PGx Testing and Personalized Medicine
•Drug exposure and clinical response
variability
• Those that do and don’t respond to a drug
•Risk for adverse events
•Genotype-specific dosing
(optimization)
•Examples:
• CYP2D6 - Cytochrome P450 2D6
• BRAF – B-Raf
• EGFR - Epidermal Growth Factor Receptor
Source: https://www.fda.gov/Drugs/ScienceResearch/ucm572698.htm
38. PGx Genotyping Solutions
•Standard TaqMan genotyping PCR
• Small panels
•PharmacoScan Array – Thermo
Fisher
• 4,627 ADME (Absorption, Distribution,
Metabolism, and Excretion) genetic
markers within 1,191 genes
•Global Screening Array – Illumina
• 13,000 ADME markers and helps
screening a diverse population
39. Challenges
• Logistics – how are
collection kits handled
• Quality Assurance – how to
insure proper sample
collection and processing at
higher volumes
•Reporting – unique to each
company and their
interpretation of the results
40. What Should Clinicians
Do?
•Work WITH the companies that do the reporting!
•Seek out a Genetic Counselor
• Trained and specializes in talking with patients
about their genetic data/reports
• Quality time in depth time with patients one on
one to discuss results
• National Society of Genetic Counselors
• https://www.nsgc.org
41. Clinical Sequencing Trend
Source: (2017, October 15) GA4GH Strikes Formal Collaborations with 15 International Genomic Data Initiatives. Retrieved from https://www.ga4gh.org/news/sAhZCeJjS96QHhVPIYwwWA.article
42. Clinically Relevant Variants
•The American College of Medical Genetics and
Genomics (ACMG) recommended that clinical
laboratories report back secondary findings
from 56 genes.
• Variants that are known to cause severe disease but
have clinically relevant actions
•Examples include
• Breast and Ovarian Cancer – BRAC1 and BRAC2
• Lynch Syndrome – MLH1, MSH2, MSH6, and PSM2
• Wilson’s Disease – ATP7B
Source: https://www.nature.com/articles/gim2016190
43. Global Initiatives
Source: (2017, October 15) GA4GH Strikes Formal Collaborations with 15 International Genomic Data Initiatives. Retrieved from https://www.ga4gh.org/news/sAhZCeJjS96QHhVPIYwwWA.article
•USA – Million Veteran
Program
•USA – All of US (1 million
participants)
https://allofus.nih.gov/
•Australian Genomics
Health Alliance
•UK – Genomics England
100,000 Genomes Project
44. Summary
•Microarrays are an important tool for large scale
genotyping studies
•Disruptive pricing allowing investigators to consider novel
large scale experiments
•New generation of Axiom and Infinium arrays enable
innovative approaches to precision medicine research
•Quality, costing, and turnaround time are crucial aspects
that ensure microarrays continue to be a critical cutting
edge tool for genomics and precision medicine research
