Pyrosequencing in Hematology

Quantify DNA methylation in a flash, like never before


Pyro Q-CpG™ is an accurate and fast analysis method for quantifying CpG methylation in epigenetic studies. Since its inception in 2002, it has established itself among leading researchers as the gold standard of DNA methylation analysis. Pyro Q-CpG has revealed correlation between DNA methylation of genes to tumor type and gene expression, measured the response to treatment with demethylating agents, as well as changes in methylation state in relation to tumorigenesis and tumor progression, genetic imprinting and exposure to environmental toxins.

The most appreciated property of Pyro Q-CpG is its highly reproducible quantification of methylation frequencies in individual consecutive CpG sites, enabling reproducible measurement of even small changes in methylation levels. The reproducibility of Pyro Q-CpG is a result of a quantitative measurement principle, inherent quality controls (QC), and few processing steps. QC is inherent because CpG sites are presented in the context of the DNA sequence, and controls for completion of the bisulfite conversion step can be integrated into the analysis process.

Quantitative DNA methylation analysis by Pyrosequencing enabled us to distinguish tumor-specific methylation from “noise” for p16, MGMT and CYGB promoters. This contrasts with previous published work using MSP, and demonstrates the significance of a quantitative rather than qualitative methylation assay. Dr. Triantafillos Liloglou, University of Liverpool Cancer Research Centre

Based on Pyrosequencing^® technology, Pyro Q-CpG analyzes and presents the individual methylation levels of multiple consecutive CpG sites in the context of the DNA sequence. Single CpG sites can be analyzed as easily as multiple sites, and methods are established for estimating global methylation.

A real-world example of methylation analysis by Pyro Q-CpG

The following example illustrates methylation analysis of the MLH1 gene. Nineteen consecutive CpG sites in the promoter region were analyzed on samples isolated from head and neck tumors. Pyrosequencing analysis performed on this 130 bp sequence takes 130 minutes for up to 96 samples. Thereafter, analysis and quality control of methylation takes 1 minute for up to 96 samples.

Using Pyro Q-CpG, regions covering multiple CpG sites can be analyzed in a single reaction. This analysis provides individual, reproducible quantification for all 19 CpG sites. In the Pyrogram® trace above, two regions, indicated in bold text in the sequence, are magnified to illustrate the individual methylation results. Methyltion levels in yellow indicate CpG sites that have not fully passed quality control.

Data on MLH1 kindly provided from Dr. Triantafillos Liloglou, University of Liverpool Cancer Research Centre,
Aintree Hospital Maxillofacial Surgery Dept.

The new Pyro Q-CpG Software makes DNA methylation analysis available for all Pyrosequencing platforms.

 Discover the awesome power of the new Pyro Q-CpG Software!  

Pyro Q-CpG™

Pyro Q-CpG from Biotage offers these advantages for methylation analysis:

• Reproducible quantification of individual, consecutive CpG sites enables discrimination of
  even small changes in methylation levels
  
  
• Versatile for a range of analyses, from single CpGs to global methylation
  
  
• Easy to communicate data: methylation levels are presented in sequence context
  
  
• Quality control: sequence context, comparison to expected methylation levels, and built-in QC for bisulfite treatment
  
• Suitable for analysis of fresh-frozen, fixed and paraffin-embedded specimens
  
  
• Robust and flexible assays, ready-made and custom-designed
  
  
• Fast turnaround: 96 samples analyzed in parallel in 1 hour after PCR
  

Pyro Q-CpG is a complete system for methylation analysis comprising instrumentation, chemistry, software, assays and analysis services. Based on Pyrosequencing^® technology, Pyro Q-CpG builds on the solid scientific and technical knowledge of our own staff, as well as routine collaborations with cancer geneticists.

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Overview
	Pyro Q-CpG: quantitative analysis of methylation in multiple CpG sites by Pyrosequencing
	White Paper 010: CpG Methylation Analysis by Pyrosequencing – Benchmarks and Application
	CpG methylation in clinical studies: utility, methods, and quality assurance
Instrumentation and Analysis Products
	Pyro Q-CpG software product note
	PyroMark MD system product note
	PyroMark MD instrument product note
PyroMark RUO tests
	PyroMark p16
	PyroMark MLH1
	PyroMark Prader-Willi/Angelman Syndromes
	PyroMark MGMT
	PyroMark LINE-1
Pyro Gold Chemistry
	Pyro Gold Chemistry
Scientific Applications
	Shaw, R. J., Hall, G. L., Lowe, D., Liloglou, T., Field, J. K., Sloan, P. and Risk, J. M. The Role of Pyrosequencing in Head and Neck Cancer Epigenetics: Correlation of Quantitative Methylation Data With Gene Expression. Arch Otolaryngol Head Neck Surg 2008. 134, 251-256.
	Kim, M., Jang, H.-R., Kim, J.-H., Noh, S.-M., Song, K.-S., Cho, J.-S., Jeong, H.-Y., Norman, J. C., Caswell, P. T., Kang, G. H., Kim, S.-Y., Yoo, H.-S. and Kim, Y. S. Epigenetic inactivation of protein kinase D1 in gastric cancer and its role in gastric cancer cell migration and invasion. Carcinogenesis 2008. 29, 629-637.
	Lee, E.-S., Issa, J.-P., Roberts, D. B., Williams, M. D., Weber, R. S., Kies, M. S. and El-Naggar, A. K. Quantitative Promoter Hypermethylation Analysis of Cancer-Related Genes in Salivary Gland Carcinomas: Comparison with Methylation-Specific PCR Technique and Clinical Significance. Clin. Cancer Res. 2008. 14, 2664-2672.
	Brakensiek K, Wingen LU, Langer F, Kreipe H, Lehmann U. Quantitative High-Resolution CpG Island Mapping with PyrosequencingTM Reveals Disease-Specific Methylation Patterns of the CDKN2B Gene in Myelodysplastic Syndrome and Myeloid Leukemia. Clin Chem. 2007 Jan;53(1):17-23
	Tost J, Jammes H, Dupont JM, Buffat C, Robert B, Mignot TM, Mondon F, Carbonne B, Simeoni U, Grange G, Kerjean A, Ferre F, Gut IG, Vaiman D. Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region. Nucleic Acids Res. 2006;34(19):5438-48
	Garcia-Manero G, Kantarjian HM, Sanchez-Gonzalez B, Yang H, Rosner G, Verstovsek S, Rytting M, Wierda WG, Ravandi F, Koller C, Xiao L, Faderl S, Estrov Z, Cortes J, O'brien S, Estey E, Bueso-Ramos C, Fiorentino J, Jabbour E, Issa JP. Phase I/II study of the combination of 5-aza-2' -deoxycytidine with valproic acid in patients with leukemia. Blood. Nov 2006; 108: 3271 - 3279
	White HE, Durston VJ, Harvey JF, Cross NC. Quantitative analysis of SNRPN(correction of SRNPN) gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome. Clin Chem. 2006 Jun;52(6):1005-13.
	Karimi M, Johansson S, Stach D, Corcoran M, Grander D, Schalling M, Bakalkin G, Lyko F, Larsson C, Ekstrom TJ. LUMA (LUminometric Methylation Assay)--a high throughput method to the analysis of genomic DNA methylation. Exp Cell Res. 2006 Jul 1;312(11):1989-95.
	Mirmohammadsadegh A, Marini A, Nambiar S, Hassan M, Tannapfel A, Ruzicka T, Hengge UR. Epigenetic silencing of the PTEN gene in melanoma. Cancer Res. 2006 Jul 1;66(13):6546-52.
	Mill J, Dempster E, Caspi A, Williams B, Moffitt T, Craig I. Evidence for monozygotic twin (MZ) discordance in methylation level at two CpG sites in the promoter region of the catechol-O-methyltransferase (COMT) gene. Am J Med Genet B Neuropsychiatr Genet. 2006 Jun 5;141(4):421-5.
	Shaw RJ, Liloglou T, Rogers SN, Brown JS, Vaughan ED, Lowe D, Field JK, Risk JM. Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer. 2006 Feb 27;94(4):561-8.
	Issa JP, Gharibyan V, Cortes J, Jelinek J, Morris G, Verstovsek S, Talpaz M, Garcia-Manero G, Kantarjian HM. Phase II Study of Low-Dose Decitabine in Patients With Chronic Myelogenous Leukemia Resistant to Imatinib Mesylate. J. Clin. Oncol., Jun 2005; 23: 3948 - 3956.
	Shiao YH, Crawford EB, Anderson LM, Patel P, Ko K. Allele-specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure. Toxicol Appl Pharmacol, Jun 2005; 205(3): 290-6.
Method Development
	Uhlmann K., Brinckmann A., Toliat M. R., Ritter H., Nürnberg P. Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis. Electrophoresis 23; 4072-9, 2002
	Colella S., Shen L., Baggerly K.A., Issa J.-P.J., Krahe R. Sensitive and quantitative universal Pyrosequencing™ methylation analysis of CpG sites. Biotechniques 35; 146-50,2003
	Tost J., Dunker J., Glynne Gut Y. Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing™. Biotechniques 35; 152-6, 2003
	Dupont JM, Tost J, Jammes H, and Gut IG. De novo quantitative bisulfite sequencing using the Pyrosequencing technology. Anal Biochem, Oct 2004; 333(1): 119-27.
	Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. Feb 2004; 32(3): e38.

Instrumentation

Pyro Q-CpG can be performed on any Pyrosequencing instrument, including

 PyroMark MD PSQ 96MA,  PSQ HS 96(A)   PyroMark ID  and  PyroMark Q24

Pyro Q-CpG Software



Pyro Q-CpG Software has been developed to address applications that benefit from high-quality and high-throughput analysis of DNA methylation. This is achieved by quantitative sequence-based analysis of multiple, consecutive CpG sites in 96 samples in parallel, enabling an individual to analyze and process many thousands of samples and CpG sites to provide statistically reliable data.

Functions that are essential for high throughput and data reliability have been incorporated into Pyro Q-CpG Software. After the Pyrosequencing run, analysis of ninety-six samples takes less than one minute, even when each sample contains 20 or more CpG sites. Another key improvement is the automatic quality control of each sample for completion of bisulfite treatment, eliminating the need for manual inspection and estimation of levels of non-converted DNA.

With the overview feature, the user can easily navigate through methylation results, mean values, methylation patterns and quality scores for each sample as well as individual CpG sites.

Pyro Q-CpG software is compatible with all existing Pyrosequencing instrument platforms. It can be installed and run in parallel with the current instrument control software.

 Take a 3 minute tour to learn more about the Pyro Q-CpG Software
 Click here to download the Pyro Q-CpG Software product note

PyroMark RUO tests for quantifying CpG methylation

PyroMark RUO tests include a number of validated tests for quantitative methylation analysis. PyroMark RUO tests provide reagents and full procedures for PCR and Pyrosequencing reactions.

 PyroMark RUO tests for analysis of CpG methylation and cancer mutations

Assay Database

Instrument users have full access to the PyroMark  on-line database containing 1,000s of Pyrosequencing assays for SNP, mutation and methylation analysis.

Optimized Chemistry


Biotage has developed  Pyro Gold, a new chemistry formulation that significantly improves the sequencing quality of nearly all Pyrosequencing assays. Optimization has particularly benefited the number of CpG sites that can be quantified in a single run.

How Pyro Q-CpG works, step-by-step

Pyro Q-CpG quantifies methylation in explicit sequence context, and is both fast and easy to perform. Assay design is flexible, since the distance from the first base to be sequenced can be varied, and therefore the primer can usually be positioned in a region free of CpG sites. In addition, there are four options for design; the assay can be performed in forward or reverse orientations on either the top or the bottom strands.

Step	Time
Typical workflow for analysis of CpG methylation for 96 samples in parallel
Bisulfite treatment	3 hours hands-on, overnight incubation
PCR amplification	2 hours
Preparation of single-stranded DNA	5 minutes hands-on, 15 minutes total
Pyrosequencing	1 minute/dispensation, typically 30 min
Calculation of methylation at each CpG site	1 minute

Typical workflow for analysis of CpG methylation for 96 samples in parallel

The approach to analyze methylation exploits one of the most powerful attributes of Pyrosequencing, namely its readout (the peaks in the Pyrogram). Unlike dideoxy (Sanger) sequencing, the peak heights produced by Pyrosequencing quantitatively report the proportions of alleles.

 

The principle of Pyrosequencing (left) and the output Pyrogram® trace (right). Bisulfite treatment and PCR converts unmethylated C to T and leaves methylated C unchanged. The partial peak heights at the fifth and sixth added nucleotides are quantitatively proportional to the ratio of C and T alleles at the illustrated CpG site.

Unmethylated C (red) and methylated C (green) are differentiated by bisulfite treatment and PCR. The ratio C:^mC at each CpG site (peaks in orange column) is measured in sequence context.
C not followed by G acts as control for the bisulfite step (blue column).

Sequence context is an important control since bisulfite-treated, PCR-amplified DNA is AT-rich, which decreases sequence variation. Pyro Q-CpG therefore guarantees that the correct sequence was analysed.

Pyro Q-CpG assays can contain an internal control for bisulfite treatment. C that is not followed by G in sequence is not methylated, and should therefore be fully converted to T by bisulfite and PCR. To confirm this, all templates should show only T and zero C in this position (blue column).

Pyro Q-CpG is practical in terms of starting material and throughput. DNA is readily analysed from both fresh frozen tissue as well as the short PCR fragment sizes that are typical of paraffin-embedded tissue, where restriction fragment analysis would be difficult. The analysis takes about 1 hour for 96 specimens in parallel, at a fraction of the cost and time of the equivalent dideoxy (Sanger) sequencing reactions.

Pyrosequencing output showing varying levels of methylation in the p16 promoter in normal tissue (top), primary tumour (middle) and metastatic lymphnodes (bottom) in head and neck cancer. Data courtesy Dr. Ralf Krahe, MD Anderson Cancer Center.
 

Download Zone

Overview
	Pyro Q-CpG: quantitative analysis of methylation in multiple CpG sites by Pyrosequencing
	White Paper 010: CpG Methylation Analysis by Pyrosequencing – Benchmarks and Application
	CpG methylation in clinical studies: utility, methods, and quality assurance
Instrumentation and Analysis Products
	Pyro Q-CpG software product note
	PyroMark MD system product note
	PyroMark MD instrument product note
PyroMark RUO tests
	PyroMark p16
	PyroMark MLH1
	PyroMark Prader-Willi/Angelman Syndromes
	PyroMark MGMT
	PyroMark LINE-1
Pyro Gold Chemistry
	Pyro Gold Chemistry
Scientific Applications
	Brakensiek K, Wingen LU, Langer F, Kreipe H, Lehmann U. Quantitative High-Resolution CpG Island Mapping with PyrosequencingTM Reveals Disease-Specific Methylation Patterns of the CDKN2B Gene in Myelodysplastic Syndrome and Myeloid Leukemia. Clin Chem. 2007 Jan;53(1):17-23
	Tost J, Jammes H, Dupont JM, Buffat C, Robert B, Mignot TM, Mondon F, Carbonne B, Simeoni U, Grange G, Kerjean A, Ferre F, Gut IG, Vaiman D. Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region. Nucleic Acids Res. 2006;34(19):5438-48
	Garcia-Manero G, Kantarjian HM, Sanchez-Gonzalez B, Yang H, Rosner G, Verstovsek S, Rytting M, Wierda WG, Ravandi F, Koller C, Xiao L, Faderl S, Estrov Z, Cortes J, O'brien S, Estey E, Bueso-Ramos C, Fiorentino J, Jabbour E, Issa JP. Phase I/II study of the combination of 5-aza-2' -deoxycytidine with valproic acid in patients with leukemia. Blood. Nov 2006; 108: 3271 - 3279
	White HE, Durston VJ, Harvey JF, Cross NC. Quantitative analysis of SNRPN(correction of SRNPN) gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome. Clin Chem. 2006 Jun;52(6):1005-13.
	Karimi M, Johansson S, Stach D, Corcoran M, Grander D, Schalling M, Bakalkin G, Lyko F, Larsson C, Ekstrom TJ. LUMA (LUminometric Methylation Assay)--a high throughput method to the analysis of genomic DNA methylation. Exp Cell Res. 2006 Jul 1;312(11):1989-95.
	Mirmohammadsadegh A, Marini A, Nambiar S, Hassan M, Tannapfel A, Ruzicka T, Hengge UR. Epigenetic silencing of the PTEN gene in melanoma. Cancer Res. 2006 Jul 1;66(13):6546-52.
	Mill J, Dempster E, Caspi A, Williams B, Moffitt T, Craig I. Evidence for monozygotic twin (MZ) discordance in methylation level at two CpG sites in the promoter region of the catechol-O-methyltransferase (COMT) gene. Am J Med Genet B Neuropsychiatr Genet. 2006 Jun 5;141(4):421-5.
	Shaw RJ, Liloglou T, Rogers SN, Brown JS, Vaughan ED, Lowe D, Field JK, Risk JM. Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer. 2006 Feb 27;94(4):561-8.
	Issa JP, Gharibyan V, Cortes J, Jelinek J, Morris G, Verstovsek S, Talpaz M, Garcia-Manero G, Kantarjian HM. Phase II Study of Low-Dose Decitabine in Patients With Chronic Myelogenous Leukemia Resistant to Imatinib Mesylate. J. Clin. Oncol., Jun 2005; 23: 3948 - 3956.
	Shiao YH, Crawford EB, Anderson LM, Patel P, Ko K. Allele-specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure. Toxicol Appl Pharmacol, Jun 2005; 205(3): 290-6.
Method Development
	Uhlmann K., Brinckmann A., Toliat M. R., Ritter H., Nürnberg P. Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis. Electrophoresis 23; 4072-9, 2002
	Colella S., Shen L., Baggerly K.A., Issa J.-P.J., Krahe R. Sensitive and quantitative universal Pyrosequencing™ methylation analysis of CpG sites. Biotechniques 35; 146-50,2003
	Tost J., Dunker J., Glynne Gut Y. Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing™. Biotechniques 35; 152-6, 2003
	Dupont JM, Tost J, Jammes H, and Gut IG. De novo quantitative bisulfite sequencing using the Pyrosequencing technology. Anal Biochem, Oct 2004; 333(1): 119-27.
	Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. Feb 2004; 32(3): e38.