pyrosequencing - university of bristol · identify if cpg in promoter region identify cgi within/...
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Pyrosequencing
Alix Groom
• high-throughput CpG methylation analysis platform
• real-time, sequence-based detection and quantification
• % methylation at multiple adjacent CpG sites
• 80-100 bases sequenced per assay • 2ug DNA analyse 3 assays/regions of interest • 24 or 96 samples/assays run at a time
Pyrosequencing
DNA extraction
Bisulphite modification
PCR Single strand template generation
Pyrosequencing
Workflow
GGTCAGTGAC/mCG
GGTUAGTGAU/mCG
C mC
U mC
Bisulphite conversion
GGTTAGTGAT/CG
U
T
mC
C
PCR amplification
Pyrosequencing analysis
G T T C A G T G A T C A G l l l l l l l l l l l l l
C mC
Bisulphite modification
R I I I I I I
Target sequence
I I I I I I F
primer annealing
I I I I I I I I I I I I
extension
copies of target sequence
Polymerase chain reaction
Biotin labelled PCR product
Anneal sequencing primer
Release single stranded DNA
Denature PCR product
Single strand generation
Capture PCR product with streptavidin beads
Pyrosequencing chemistry
T A G T A G G
A T C A T
3’
3’ 5’
5’
Polymerase Polymerase
DNA(n) + dNTP DNA(n+1) + PPi
Sulfurylase
APS + PPi ATP
Luciferase
ATP Light
Light
Time
Apyrase dNTP dNDP + dNMP + phosphate
Apyrase ATP ADP + AMP + phosphate
G T A G G -
G T A G G
Nucleotide sequence
Nucleotide added
DNA polymerase ATP sulfurylase
Luciferase Apyrase
APS Luciferin
Bisulphite modification
PCR Single strand template generation
Pyrosequencing
Day 1
Day 2
Day 2
DNA-reagent incubation 3hrs
sample preparation 15min-1hr
column purification 30min-1hr
PCR set up 30min-1hr
PCR cycles 1.5hr
agarose gel 1hr
sample prep 30min-1hr
Pyrosequencing run 10min-1.5hr
Day 3 Day 3
Pyrosequencing timeline
Pyrosequencing applications
• Gene specific methylation analysis identified target loci through gene expression studies literature search methylation arrays etc
• Global methylation analysis methylation of repetitive elements LUMA
Case study
Illumina 27K/450K top hit
Pyrosequencing VeraCode Sequenom
verify
Check no SNPs in probe which DNA strand CpG site is measured
Case study identify if CpG in promoter region
identify CGI within/ adjacent to promoter
capture sequence 4000bp flanking
CGI
identify TFBM that contain CpG
select CpG of interest
identify if CpG in promoter region
identify CGI within/ adjacent to promoter
capture sequence 4000bp flanking
CGI
identify TFBM that contain CpG
select CpG of interest
• genomatix Gene2Promoter software
Case study promoter region
identify if CpG in promoter region
identify CGI within/ adjacent to promoter
capture sequence 4000bp flanking
CGI
identify TFBM that contain CpG
select CpG of interest
• genomatix Gene2Promoter software
Case study promoter region
identify if CpG in promoter region
identify CGI within/ adjacent to promoter
capture sequence 4000bp flanking
CGI
identify TFBM that contain CpG
select CpG of interest
• genomatix Gene2Promoter software
Case study promoter region
identify if CpG in promoter region
identify CGI within/ adjacent to promoter
capture sequence 4000bp flanking
CGI
identify TFBM that contain CpG
select CpG of interest
• genomatix Gene2Promoter software
Case studypromoter region
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics • CpG Island explorer
Case studyCpG Island
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
Case studyCpG Island
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
Case studyCpG Island
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
Case studyCpG Island
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
Case studyCpG Island
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
• CGI Chr10:48 827 593-48 828 126 • 4000bp downstream 48 823 593 • 4000bp upstream 48 832 126
Case studysequence capture
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
Chr10: 48823593 - 48832126
Case studysequence capture
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
Case studysequence capture
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
Case studysequence capture
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
Case studysequence capture
CpG Shelf CpG Shore CpG Island
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
• UCSC Genome Bioinformatics http://genome.ucsc.edu/
Case studysequence capture
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
Case studytranscription factor binding
HNF1 GATA
TTGTACTAACGATATGCCATGCTA TTGTACTAACGATATGCCATGCTA
HNF1 GATA
module
• UCSC TFBS single binding factor information • JASPAR http://jaspar.cgb.ki.se • TRANSFAC http://www.gene-regulation.com/pub/databases.html TRANSCompel • Genomatix ModelInspector
• TFBM defined 2+ TFBS in defined order and orientation
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
Case studytranscription factor binding module
Genomatix
http://www.genomatix.de/
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
Case studytranscription factor binding module
Genomatix
http://www.genomatix.de/
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
Case studytranscription factor binding module
Genomatix
http://www.genomatix.de/
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
Case studytranscription factor binding module
Genomatix
http://www.genomatix.de/
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
Case studytranscription factor binding module
identify if CpG in promoter region
capture sequence 4000bp flanking
CGI
select CpG of interest
identify CGI within/ adjacent to promoter
identify TFBM that contain CpG
Case study
• If analysing specific CpG can capture adjacent CpGs • Do you want to analyse CGI
CpG Shore CpG Shelf CpG Open Sea
Case studyPSQ design software
• Paste in bisulphite modified sequence of interest flanked by ~300bp • Select target CpG • Maximum amplicon length ~600bp
• Ensure primers do not cover SNPs or CpGs
Case studyPSQ design software
forward primer
sequencing primer
reverse primer
Case studyPyroMark CpG assays
• 84, 000+ predesigned assays 30,000+ human assays 30,000+ mouse assays 24,000+ rat assays http://www.qiagen.com/products/pyromarkcpgassays.aspx
• Level of methylation, accuracy within ~ 5% exclude assays with <5% or >95% methylation • No preferential amplification of unmethylated or methylated DNA White HE, Clinical Chemistry 52:6 1005-1013, 2006
• Bisulphite conversion is completed
Pyrosequencing “checks”
Pyrosequencing run
• Run time dependent on sequence length, 10min-1.5hr
Pyrosequencing analysis
Pyrosequencing analysis
• Samples run in duplicate are within 5% • 0% and 100% controls are comparable between plates • inter/intraplate replicates are comparable • negative DNA control no signal
Pyrosequencingsummary
• high-throughput CpG methylation analysis platform
• real-time, sequence-based detection and quantification
• % methylation at multiple adjacent CpG sites
• genotyping
References
• Helen E. White, Clinical Chemistry 52:6 1005–1013 (2006) Quantitative Analysis of SRNPN Gene Methylation by Pyrosequencing as a Diagnostic Test for Prader–Willi Syndrome and Angelman Syndrome • http://www.pyrosequencing.com/ • http://www.qiagen.com/products/bytechnology/pyrosequencing • UCSC Genome Bioinformatics http://genome.ucsc.edu/ • Genomatix http://www.genomatix.de/
Pyrosequencing
Alix Groom