illumina veracode - bristol
TRANSCRIPT
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