General Principles of Intellectual Property: Concepts of Intellectual Proper...
High throughput genotyping
1. High throughput SNP genotyping
Cosentino Cristian, PhD
Genomics and Bioinformatics unit
Filarete Foundation – Milan
cosentia@gmail.com
2. 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)
3. 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
4. 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
9. Sample preparation
DNA arrival DNA extraction
Sample
validation Move vials into 96
well plates
Sample
preparation
DNA quantification
Genotyping
DNA dilution
RawData
evaluation
Quality control on gel
GWA analysis
Move to new fullfilled
plate
10. High throughput sample management
Tecan Liquid handler
DNA extraction
DNA arrival
Move vials into 96
Sample
well plates
validation
Sample
DNA quantification
preparation
Genotyping DNA dilution
RawData
evaluation Quality control on gel
GWA analysis
Move to new fullfilled Usage:
plate 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)
11. High throughput sample management
Multi-sample spectrophotometer
DNA extraction
DNA arrival
Move vials into 96
Sample
well plates
validation
Sample
DNA quantification
preparation
Genotyping DNA dilution
RawData
evaluation Quality control on gel
GWA analysis Usage:
Move to new fullfilled
plate 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
12. High throughput sample management
Pre-casted agarose gel
DNA extraction
DNA arrival
Move vials into 96
Sample
well plates
validation
Sample
DNA quantification
preparation
Genotyping DNA dilution
RawData
evaluation Quality control on gel
GWA analysis Usage:
Move to new fullfilled
plate 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
14. Single base extension
DNA arrival
Single base extension
Sample Markers: ddA, ddT, ddC, ddG
validation
Sample
preparation
Genotyping
RawData
evaluation
GWA analysis
