BLOOD $ URINE COLLECTION Set of blood collection tubes 1 x 4.5 ml. Citrate vacutainer Citrated plasma 2 x 9 ml. EDTA vacutainer DNA isolation and EDTA-plasma 1 x 2 ml. EDTA vacutainer Measurement of HbA1c, hemoglobin and hematocrit 2 x 9 ml. Heparin vacutainer RNA isolation Urine collection set 2 x vacutainers, 1 x adaptor Collection of urine 1 x 9 ml. Heparin vacutainer Isolation of Leucocytes and heparin plasma

BLOOD & URINE COLLECTION 2 x 9 ml. EDTA vacutainer 2 x 9 ml. Heparin vacutainer 1 x 9 ml. Heparin vacutainer 1 x 4.5 ml. Citrate vacutainer 2 x 10 ml. Urine 1 x 2 ml. EDTA vacutainer Store in melting ice during transport Store in melting ice during transport Tube A: Freeze within 1 hr. Store in dry-ice during transport Tube B: Add challenger within 1 hr. Store at 37°C during transport Store at room temperature during transport

EDTA: DNA & PLASMA BLOOD HANDLING 2 x 9 ml. EDTA vacutainer Store in melting ice during transport Within 6 hr. Centrifuge: 20 min. 2000 G and 4°C Vacutainer with red cells, buffy- coat and rest plasma Collect plasma in plastic tube Vortex shortly <1 hr RT Store at -20 °C Division of plasma 18 Subsamples of ~500 µl <1 hr RT Snap-freezen Store at <-30 °C Methanol / Dry-ice

Heparine: RNA (rest & challenge) BLOOD HANDLING 2 x 9 ml. Heparine vacutainer TUBE A TUBE B <1 hr RT <1 hr RT Add challenger 3 subsamples of ~ 3 ml Store at 37°C during transport Snap-freezen After 6 hr. (exactly) Methanol / Dry-ice 3 subsamples of ~ 3 ml Store in dry-ice during transport Snap-freezen Methanol / Dry-ice After arrival at TNO Store at<-60 °C Store at<-60 °C

** Year 2004 to Nov only

Array Based Genotyping Costs must reduce further The Sentrix Whole-Genome Genotyping BeadChip 100,000 SNPs 25,000 in transcripts 70,000 within 10kb of exons Affymetrix 100K SNP Chips

AGRF Affymetrix Chip Genotyping Concordance and fail rates Concordance comparison of samples with SNP fail rate of < 3% Samples tested were an MZ twin pair, plus parents (S3 and S4). Genomic, Buccal and MDA Genomic replicates were tested for each individual. Sample MZ 2 Buccal was tested twice. Fail - no call for one or both samples

Association Analysis Sharing between unrelated individuals Disease alleles originate in common ancestor High resolution Recombination since common ancestor Large number of independent tests Powerful if assumptions are met Same disease haplotype shared by many patients Sensitive to population structure

Single Nucleotide Polymorphisms (SNP) GGCTTCAGAATGGCC GGCTTCAAAATGGCC Single base changes Human SNPs = 9,856,125 - Validated SNPs 4,540,241 Frequency ~ 1 every 400 bp Can cause functional changes

QIMR’s Sequenom MassARRAY Installation (CCRC-E floor)

Multiplex Analysis * A G * A G * T C * A G * C T

SNP Genotyping Minimum Finished Genotypes (>99%) Quality of DNA Measure concentrations Dispense in large volumes Quality of Assays Even peak heights Test for Hardy-Weinberg equilibrium Analysis of SNP data is particularly sensitive to assay problems Genotype failures are not random Heterozygous individuals fail most often all SNP typing platforms Error frequency of 0.11% 3268 DNA samples typed twice 159 pairs of MZ twins - No discordant genotypes

Twelve-Plex Genotyping

Whole Genome Association Use DNA pooling to greatly reduce amount of genotyping Possible now but reduced power Use haplotypes to reduce number of SNPs that have to be genotyped Haplotype blocks – HapMap project Massive parallel genotyping Costs must reduce further