high throughput genotyping
TRANSCRIPT
Cosentino Cristian, PhD
Genomics and Bioinformatics unit
Filarete Foundation – Milan
High throughput SNP genotyping
Illumina genotyping
• Illumina BeadArray technology
• Simultaneous genotyping of thousand to milion of loci
• Entire genome accessible to analysis
• Low amount of gDNA required (250 – 750 ng)
• Automation of the protocol
• Randomly assembled arrays (decoding process)
High throughput
The laboratory is divided in:
1. WetLab: working on biological samples
(Biotechnologists, Technicians)
• Pre-PCR area
• Post-PCR area
2. DryLab: working on data analysis
(Bioinformaticians, IT systemists, Clinicians)
[WetLab] Samples management for Genotyping:
1. Sample validation
2. Sample preparation
3. Genotyping
4. RawData evaluation
Genotyping protocols
GoldenGate:
96 samples/array and thousand of SNPs simultaneously assayed; completely cutomizable
Infinium:
2-12 samples/chip and hundred of thousand of SNPs simultaneously assayed; customizable
up to 20-30.000 ca. SNPs per sample
GoldenGate protocol Infinium protocol
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Optic fiber
Solid array
Gold
engat
e w
ork
flow
Infi
niu
m w
ork
flow
PCR-based
amplification
Whole-genome
amplification
Infinium overview
Sample validation
Sample preparation
Genotyping
RawData evaluation
DNA arrival
GWA analysis
Sample validation
SAMPLE arrival
(Plate)
SAMPLE present
DNA_ID missing
Consistency check
DNA_ID arrival
(List)
SAMPLE missing
DNA_ID present
Double DNA_ID
Batch validation
Wait for sender’s
feedback- Errors -
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
Sample preparation
Move vials into 96
well plates
DNA quantification
Quality control on gel
Move to new fullfilled
plate
DNA dilution
DNA extraction
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
High throughput sample management
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
Move vials into 96
well plates
DNA quantification
Quality control on gel
Move to new fullfilled
plate
DNA dilution
DNA extraction
Tecan Liquid handler
Usage:
Vial-to-plate and Plate-to-plate sample movement
Normalization of the sample concentration
Advantages:
1. Cost-saving (30’ x 2 operators vs 20’ x 1 operator/plate)
2. Tracking of the sample movement (personal responsability)
High throughput sample management
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
Move vials into 96
well plates
DNA quantification
Quality control on gel
Move to new fullfilled
plate
DNA dilution
DNA extraction
Multi-sample spectrophotometer
Usage:
Nucleic acid quantification
Advantages:
1. 16 samples/run assayed
2. Small sample volume used
3. Quantification in standard routine
4. Connection to the liquid handler normalization software
High throughput sample management
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
Move vials into 96
well plates
DNA quantification
Quality control on gel
Move to new fullfilled
plate
DNA dilution
DNA extraction
Pre-casted agarose gel
Usage:
Screening of the DNA integrity
Advantages:
1. 48 samples/run assayed in 20’
2. Standarsd screening conditions
3. System copatible with TECAN liquid handler
4. Barcode ID for easy data collection
Genotyping
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
Single base extension
Single base extension
Markers: ddA, ddT, ddC, ddGSample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
Xstain
BeadType
20-30xSample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
Genotype calling
AA BB AB Genotype
Detection
Bead color
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
Scanning
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
High throughput genotyping
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
BeadScan
iScan
1. Autoloader for automatic non-stop
beadchip scanning
2. Data directly stored to the back-up utility
3. 1M-duo chip/3h scan
4. Max 22 chip/run
1. Autoloader for automatic non-stop
beadchip scanning
2. Data directly stored to the back-up utility
3. 1M-duo chip/40’ scan
4. Max 44 chip/run
High throughput genotyping
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
TECAN liquid handler
1. Automatic management of loading and
Xstain Infinium steps
2. Tracking of the procedures
3. Max 96 chip/working week
Genotype calling
SNP calls
(CallRate >0.95)
Data Storage
(10 -100 Gb/project)
ManifestData
GenomeStudio
FinalReport
(Genotypes)
DryLab GWA analysis
RawData
Matrix:
columns (param.)
x raws (#DNA x
#SNPs)
GWAS:
Genotypes x
Phenotypes
Reference
Clusters
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
GenomeStudio
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
High throughput RawData evaluation
Sample
validation
Sample
preparation
Genotyping
RawData
evaluation
DNA arrival
GWA analysis
Windows server
Usage:
Genotype calling by the Illumina GenomeStudio software
System:
• Intel Xeon X5450 3.00 GHz
• 8 x CPU
• 16 Gb RAM
• 3 Tb data storage capacity
Infinium beadchips roadmap
Cosentino Cristian, PhD
Genomics and Bioinformatics unit
Filarete Foundation – Milan