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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
- 5. GoldenGate protocol Infinium protocol ... ... ... ... ... ... ... ... ... ... ... ... ... Optic fiber Solid array
- 6. Whole-genome amplification PCR-based amplification Goldengate workflow Infinium workflow
- 7. Infinium overview DNA arrival Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
- 8. Sample validation SAMPLE arrival DNA_ID arrival (Plate) (List) DNA arrival Sample Consistency check validation Sample Wait for sender’s - Errors - preparation feedback SAMPLE present DNA_ID missing Genotyping SAMPLE missing RawData DNA_ID present evaluation Double DNA_ID GWA analysis Batch validation
- 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
- 13. Genotyping DNA arrival Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
- 14. Single base extension DNA arrival Single base extension Sample Markers: ddA, ddT, ddC, ddG validation Sample preparation Genotyping RawData evaluation GWA analysis
- 15. Xstain DNA arrival BeadType 20-30x Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
- 16. Genotype calling DNA arrival Sample validation Sample preparation Genotyping RawData evaluation Detection GWA analysis Bead color AA BB AB Genotype
- 17. Scanning DNA arrival Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
- 18. High throughput genotyping BeadScan DNA arrival 1. Autoloader for automatic non-stop beadchip scanning 2. Data directly stored to the back-up utility Sample 3. 1M-duo chip/3h scan validation 4. Max 22 chip/run Sample preparation Genotyping RawData iScan evaluation 1. Autoloader for automatic non-stop beadchip scanning GWA analysis 2. Data directly stored to the back-up utility 3. 1M-duo chip/40’ scan 4. Max 44 chip/run
- 19. High throughput genotyping DNA arrival Sample TECAN liquid handler validation 1. Automatic management of loading and Sample Xstain Infinium steps preparation 2. Tracking of the procedures 3. Max 96 chip/working week Genotyping RawData evaluation GWA analysis
- 20. Genotype calling DNA arrival Reference RawData GenomeStudio Clusters Sample validation ManifestData Sample SNP calls preparation (CallRate >0.95) FinalReport Matrix: Genotyping columns (param.) (Genotypes) x raws (#DNA x Data Storage #SNPs) RawData evaluation (10 -100 Gb/project) GWA analysis GWAS: DryLab GWA analysis Genotypes x Phenotypes
- 21. GenomeStudio DNA arrival Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
- 22. High throughput RawData evaluation Windows server DNA arrival Sample validation Sample preparation Genotyping RawData evaluation Usage: GWA analysis 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
- 24. Cosentino Cristian, PhD Genomics and Bioinformatics unit Filarete Foundation – Milan cosentia@gmail.com