Pyrosequencing - University of Bristol · identify if CpG in promoter region identify CGI within/...

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