High throughput SNP genotyping


               Cosentino Cristian, PhD
               Genomics and Bioinformatics unit
  ...
Illumina genotyping




•   Illumina BeadArray technology
•   Simultaneous genotyping of thousand to milion of loci
•   En...
High throughput



The laboratory is divided in:
1. WetLab: working on biological samples
   (Biotechnologists, Technician...
Genotyping protocols
GoldenGate:
96 samples/array and thousand of SNPs simultaneously assayed; completely cutomizable

Inf...
GoldenGate protocol   Infinium protocol




                                                      ...
                    ...
Whole-genome
                                                          amplification
                      PCR-based
     ...
Infinium overview



      DNA arrival



   Sample validation



   Sample preparation



      Genotyping



   RawData ...
Sample validation



                                          SAMPLE arrival                   DNA_ID arrival
           ...
Sample preparation




DNA arrival               DNA extraction


  Sample
 validation             Move vials into 96
    ...
High throughput sample management

                                                       Tecan Liquid handler

          ...
High throughput sample management

                                          Multi-sample spectrophotometer

             ...
High throughput sample management

                                                   Pre-casted agarose gel

            ...
Genotyping




DNA arrival


  Sample
 validation

   Sample
 preparation


 Genotyping


  RawData
 evaluation


GWA anal...
Single base extension




DNA arrival
                                  Single base extension
  Sample                    ...
Xstain




DNA arrival    BeadType
                20-30x
  Sample
 validation

   Sample
 preparation


 Genotyping


  R...
Genotype calling




DNA arrival


  Sample
 validation

   Sample
 preparation


 Genotyping


  RawData
 evaluation     ...
Scanning




DNA arrival


  Sample
 validation

   Sample
 preparation


 Genotyping


  RawData
 evaluation


GWA analys...
High throughput genotyping


                        BeadScan

DNA arrival    1. Autoloader for automatic non-stop
       ...
High throughput genotyping




DNA arrival


  Sample
               TECAN liquid handler
 validation
               1. Au...
Genotype calling




DNA arrival
               Reference     RawData         GenomeStudio
                Clusters
  Samp...
GenomeStudio




DNA arrival


  Sample
 validation

   Sample
 preparation


 Genotyping


  RawData
 evaluation


GWA an...
High throughput RawData evaluation

                                  Windows server

DNA arrival


  Sample
 validation

...
Infinium beadchips roadmap
Cosentino Cristian, PhD
Genomics and Bioinformatics unit
Filarete Foundation – Milan
cosentia@gmail.com
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High throughput genotyping

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High throughput genotyping

  1. 1. High throughput SNP genotyping Cosentino Cristian, PhD Genomics and Bioinformatics unit Filarete Foundation – Milan cosentia@gmail.com
  2. 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. 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. 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. 5. GoldenGate protocol Infinium protocol ... ... ... ... ... ... ... ... ... ... ... ... ... Optic fiber Solid array
  6. 6. Whole-genome amplification PCR-based amplification Goldengate workflow Infinium workflow
  7. 7. Infinium overview DNA arrival Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
  8. 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. 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. 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. 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. 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. 13. Genotyping DNA arrival Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
  14. 14. Single base extension DNA arrival Single base extension Sample Markers: ddA, ddT, ddC, ddG validation Sample preparation Genotyping RawData evaluation GWA analysis
  15. 15. Xstain DNA arrival BeadType 20-30x Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
  16. 16. Genotype calling DNA arrival Sample validation Sample preparation Genotyping RawData evaluation Detection GWA analysis Bead color AA BB AB Genotype
  17. 17. Scanning DNA arrival Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
  18. 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. 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. 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. 21. GenomeStudio DNA arrival Sample validation Sample preparation Genotyping RawData evaluation GWA analysis
  22. 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
  23. 23. Infinium beadchips roadmap
  24. 24. Cosentino Cristian, PhD Genomics and Bioinformatics unit Filarete Foundation – Milan cosentia@gmail.com
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