Illumina VeraCode

Alix Groom

illumina BeadXpress Reader

• Custom low to mid plex genotyping

48-384 plex

• Custom low to mid plex methylation analysis

96-384 plex

• SNP screening

• Protein screening

illumina BeadXpress Reader

• Automated fluidics and multi-laser imaging device

• VeraCode technology

– Holographic microbeads for customisable tracking

• High throughput

– 80 samples/hr for 96-plex assay

• Robust

– Up to 300 data points for each analyte

VeraCode Technology

CODE IMAGE

1 0 1 1 0 1 0 1 0 0 0 0 1 0 1

BINARY CODE

DECIMAL CODE = 41133

CODE GENERATED BY CREATING EMBEDDED HOLOGRAPHIC DIFFRACTIVE ELEMENTS

• Glass Surface of Beads Ideal for Bioassays

• High Density Codes Easily Imprinted (24 bit)

• Virtually Unlimited Multiplexing

CONVENTIONAL CCD CAMERA

BEAD

“READING” BEAM

• GoldenGate Genotyping • GoldenGate Methylation

VeraCodeUniversal Capture bead

• Multiplexing achieved through pooling bead types • VeraCode bead types distributed into wells • Maximum 384 bead types

VeraCodeUniversal Capture bead

DNA methylation workflow Genomic DNA (500ng for two uses)

Methylated locus

CG CpG locus

Bisulphite conversion

Add DNA to oligonucleotides, hybridise

CG G A

P2

AS02 5’

P1 AS01 5’

P3 3’ LSO1

P3 3’ LSO2

Extend, ligate, clean up

CG P2

AS02 5’ G

P3 3’ LSO2

Universal PCR cycle at up to 384 plex

P2

P3

Hybridise the VeraCode BeadPlate

methylated semimethylated

unmethylated

Wash the VeraCode BeadPlate

Scan the VeraCode BeadPlate

Methylation output β value 0 = unmethylated 1 = fully methylated

• Includes software to analyse data – Genome Studio

• Export data as .csv files or in report format e.g. heat maps, scatter plots

• Methylation levels given as β value: 0=0% methylation 1=100% methylation

Data Outputs

Make BCS (bisulphite converted

single use DNA)

Start bisulphite conversion

Make BCD (bisulphite converted

DNA)

Precipitate BCS

Resuspend SUD

Make ASE allele specific

extension

Add MEL master mix for

extension and ligation

Make PCR

Inoc PCR

Cycle PCR

Bind PCR

Make INT VBP (intermediate plate

for VeraCode BeadPlate

Hyb VBP (hybridise VeraCode

BeadPlate)

Wash VBP

Image VBP

Day 1 Day 2 Day 3

Pre-PCR

Post-PCR

Case Study

• congenital cardiac malformations are the most common of all human congenital abnormalities • development of the fetal heart is orchestrated by transcriptional regulation of key developmental genes • genetic lesions in some of these genes have been shown to cause cardiac malformations • environmental factors have also been implicated in transcriptional perturbation in cardiac development and subsequent cardiac malformations • epigenetic mechanisms may mediate the relationship between teratogenic exposure and the development of cardiac malformations

AIM: Identify DNA methylation patterns in human heart development Rosa Spencer, Susan Lindsay, Laura Yates, Caroline Relton

identify candidate genes

identify CpG of interest CGI, CpG shore/shelf

TFBM

submit CpGs for scoring

design final panel

VeraCode methylation protocol

Case Studyworkflow

Case Studyidentify CpGs

• Target gene approach literature search gene expression data set

15 14

• Identify promoter region • Identify CGI within/adjacent to promoter • Capture sequence 4000bp upstream, 4000bp downstream CGI • Identify TFBM that contain CpG within their motif

• Submit sequence 60bp flanking CpG

• Identify CpGs score > 0.8 ~7% success rate

Case StudyVeraCode scoring

Case Studypanel design

• Exclude CpGs that contain 1+ CpGs within 60bp flanking site

42%

58%

CpG loci

Shore

Island

AARS ACTC1 AP2A2 BMP4 CAND1 CEP192 CHD7 COL18A1 EXOSC1 GATA4 IGF2 ISL1 MEF2C MEST MYLK3 NKX2-5 NOP56 PAX3 POP1 PTPN11 PTPN13 RCBTB1 SF3A1 SNRNP48 SNRNP70 TBX20 TBX5 TGFB1 TPX2 VEGFA WNT5A

Total CpG per gene

• 96 CpGs in 31 genes • 3-5 CpGs per gene

• 134 fetal heart tissue DNA, 104 placental DNA • Small sample subset duplicated across plates to assess batch effect

Case Studyraw data

Illumina standard controls Allele specific extension Extension gap Bisulphite conversion 1st hybridisation Gender controls 2nd hybridisation Negative controls Contamination detection

Case Studyquality control

• Exclude

Observations detection p value > 0.05 Probes with failure rate > 20% Individual samples failure rate > 20%

• Check for batch effect intra/inter plate duplicates

Case Studydata cleaning

Case Studyresults

0.2

.4.6

.81

Meth

yla

tio

n b

eta

va

lue (

%)

snrn

p7

0_

p1

snrn

p7

0_

s1

tbx5_

p1

tbx5_

p2

tbx5_

p3

tbx5_

p4

tbx5_

s1

tbx20

_p

1

tbx20

_s1

tgfb

1_

p1

tgfb

1_

p2

tgfb

1_

p3

tgfb

1_

s1

tgfb

1_

s2

tpx2_

p1

tpx2_

p2

tpx2_

p3

tpx2_

p4

tpx2_

p5

veg

fa_p

1

wn

t5a_

1_

p1

wn

t5a_

1_

p2

wn

t5a_

1_

s1

wn

t5a_

2_

p1

wn

t5a_

2_

s1

CpG sites

7 to 8 weeks 9 to 11 weeks 12 to 14 weeks

In heart tissue stratified for developmental group

Distribution of methylation

Summary

• High throughput • Can custom design your panel • 96-384 plex • Scoring success rate low • CpGs within CGIs low scores • Minimum initial order of 480 samples

References

• http://www.illumina.com/technology/veracode_technology.ilmn • http://www.illumina.com/software/genomestudio_software.ilmn

Illumina VeraCode

Alix Groom