high throughput genotyping

Cosentino Cristian, PhD

Genomics and Bioinformatics unit

Filarete Foundation – Milan

cosentia@gmail.com

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

...

...

...

...

... ... ... ... ... ... ... ... ...

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

cosentia@gmail.com