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Characterization of HPV DNA methylation of contiguous CpG sites by bisulfite treatment and massively parallel sequencing-the FRAGMENT approach.

Sun C, McAndrew T, Smith BC, Chen Z, Frimer M, Burk RD - Front Genet (2014)

Bottom Line: DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides and increased methylation across the HPV16 genome is strongly associated with ICC development.Moreover, it provides additional information on methylation "haplotypes." In the current study, we chose 12 random samples, amplified multiple segments in the HPV16 bisulfite treated genome with specific barcodes, inspected the methylation ratio at 31 CpG sites for all samples using Illumina sequencing, and compared the results with quantitative pyrosequencing.In summary, the advantages of Next Gen sequencing compared to pyrosequencing for HPV genome methylation analyses include higher throughput, increased resolution, and improved efficiency of time and resources.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Albert Einstein College of Medicine Bronx, NY, USA.

ABSTRACT
Invasive cervix cancer (ICC) is the third most common malignant tumor in women and human papillomavirus 16 (HPV16) causes more than 50% of ICC. DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides and increased methylation across the HPV16 genome is strongly associated with ICC development. Next generation (Next Gen) sequencing has been proposed as a novel approach to determine DNA methylation. However, utilization of this method to survey CpG methylation in the HPV16 genome is not well described. Moreover, it provides additional information on methylation "haplotypes." In the current study, we chose 12 random samples, amplified multiple segments in the HPV16 bisulfite treated genome with specific barcodes, inspected the methylation ratio at 31 CpG sites for all samples using Illumina sequencing, and compared the results with quantitative pyrosequencing. Most of the CpG sites were highly consistent between the two approaches (overall correlation, r = 0.92), thus verifying that Next Gen sequencing is an accurate and convenient method to survey HPV16 methylation and thus can be used in clinical samples for risk assessment. Moreover, the CpG methylation patterns (methylation haplotypes) in single molecules identified an excess of complete-and non-methylated molecules and a substantial amount of partial-methylated ones, thus indicating a complex dynamic for the mechanisms of HPV16 CpG methylation. In summary, the advantages of Next Gen sequencing compared to pyrosequencing for HPV genome methylation analyses include higher throughput, increased resolution, and improved efficiency of time and resources.

No MeSH data available.


Related in: MedlinePlus

The summary correlation of 27 CpG methylation sites between pyrosequencing and Next Gen sequencing. The x-axis is percent methylation by pyrosequencing and the y-axis is percent methylation by Next Gen sequencing. Each CpG site is plotted for each subject.
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Figure 1: The summary correlation of 27 CpG methylation sites between pyrosequencing and Next Gen sequencing. The x-axis is percent methylation by pyrosequencing and the y-axis is percent methylation by Next Gen sequencing. Each CpG site is plotted for each subject.

Mentions: Since L1, L2, and E2 ORF regions showed differential methylation among disease groups (Mirabello et al., 2012), these three regions and the most significant CpG sites within them were chosen for the current study. Primers for PCR were designed using MethPrimer (Li and Dahiya, 2002) (http://www.urogene.org/methprimer/index1.html). In total, 3 segments in L1, 4 in L2, and 1 in E2 were included in the current study (Table 1A), and in total, 31 CpG sites were surveyed. Each primer was labeled by a unique barcode and 5′ and 3′ padding sequence (Table 1B) and synthesized by Integrated DNA Technologies (IDT, Coralville, IA). A map of all 112 CpG sites in the HPV16 7906 bp reference genome is shown in Figure 1 in the review by Clarke et al. (2012).


Characterization of HPV DNA methylation of contiguous CpG sites by bisulfite treatment and massively parallel sequencing-the FRAGMENT approach.

Sun C, McAndrew T, Smith BC, Chen Z, Frimer M, Burk RD - Front Genet (2014)

The summary correlation of 27 CpG methylation sites between pyrosequencing and Next Gen sequencing. The x-axis is percent methylation by pyrosequencing and the y-axis is percent methylation by Next Gen sequencing. Each CpG site is plotted for each subject.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042685&req=5

Figure 1: The summary correlation of 27 CpG methylation sites between pyrosequencing and Next Gen sequencing. The x-axis is percent methylation by pyrosequencing and the y-axis is percent methylation by Next Gen sequencing. Each CpG site is plotted for each subject.
Mentions: Since L1, L2, and E2 ORF regions showed differential methylation among disease groups (Mirabello et al., 2012), these three regions and the most significant CpG sites within them were chosen for the current study. Primers for PCR were designed using MethPrimer (Li and Dahiya, 2002) (http://www.urogene.org/methprimer/index1.html). In total, 3 segments in L1, 4 in L2, and 1 in E2 were included in the current study (Table 1A), and in total, 31 CpG sites were surveyed. Each primer was labeled by a unique barcode and 5′ and 3′ padding sequence (Table 1B) and synthesized by Integrated DNA Technologies (IDT, Coralville, IA). A map of all 112 CpG sites in the HPV16 7906 bp reference genome is shown in Figure 1 in the review by Clarke et al. (2012).

Bottom Line: DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides and increased methylation across the HPV16 genome is strongly associated with ICC development.Moreover, it provides additional information on methylation "haplotypes." In the current study, we chose 12 random samples, amplified multiple segments in the HPV16 bisulfite treated genome with specific barcodes, inspected the methylation ratio at 31 CpG sites for all samples using Illumina sequencing, and compared the results with quantitative pyrosequencing.In summary, the advantages of Next Gen sequencing compared to pyrosequencing for HPV genome methylation analyses include higher throughput, increased resolution, and improved efficiency of time and resources.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Albert Einstein College of Medicine Bronx, NY, USA.

ABSTRACT
Invasive cervix cancer (ICC) is the third most common malignant tumor in women and human papillomavirus 16 (HPV16) causes more than 50% of ICC. DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides and increased methylation across the HPV16 genome is strongly associated with ICC development. Next generation (Next Gen) sequencing has been proposed as a novel approach to determine DNA methylation. However, utilization of this method to survey CpG methylation in the HPV16 genome is not well described. Moreover, it provides additional information on methylation "haplotypes." In the current study, we chose 12 random samples, amplified multiple segments in the HPV16 bisulfite treated genome with specific barcodes, inspected the methylation ratio at 31 CpG sites for all samples using Illumina sequencing, and compared the results with quantitative pyrosequencing. Most of the CpG sites were highly consistent between the two approaches (overall correlation, r = 0.92), thus verifying that Next Gen sequencing is an accurate and convenient method to survey HPV16 methylation and thus can be used in clinical samples for risk assessment. Moreover, the CpG methylation patterns (methylation haplotypes) in single molecules identified an excess of complete-and non-methylated molecules and a substantial amount of partial-methylated ones, thus indicating a complex dynamic for the mechanisms of HPV16 CpG methylation. In summary, the advantages of Next Gen sequencing compared to pyrosequencing for HPV genome methylation analyses include higher throughput, increased resolution, and improved efficiency of time and resources.

No MeSH data available.


Related in: MedlinePlus