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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 correlation of 27 individual CpG methylation sites between pyrosequencing and Next Gen sequencing. Each plot displays one CpG site and the location is indicated at the top. The x-axis indicates Illumina sequencing, the y-axis pyrosequencing for percent methylation.
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Figure 2: The correlation of 27 individual CpG methylation sites between pyrosequencing and Next Gen sequencing. Each plot displays one CpG site and the location is indicated at the top. The x-axis indicates Illumina sequencing, the y-axis pyrosequencing for percent methylation.

Mentions: In total, 192.2 million reads 95 bp long were obtained from NG sequencing and 53.4 million (27.8%) possessed an average Phred score above 20 and were used for this analysis (Ewing and Green, 1998). 41.7 million reads (78.1%) were observed to contain one of the incorporated barcodes without mismatch and assigned to a corresponding sample for further analysis. Except one segment in the L2 gene, most segments included ~4–8 million reads (Figure S1A). For each sample, the read count varied from 2 to 6 million (Figure S1B). Most CpG sites (21/27) were covered by 0.6 to 1.8 million reads (Figure S1C), in total. Although three fragments did not amplify as robustly and had less reads (i.e., L1_2, L2_2, and L2_5 assays containing CpG sites 7034, 7091, 7136, 7145; 4427, 4437, 4441; and 5378, respectively), the correlation between CpG sites within these fragments between the PSQ and NGS assays had reasonable agreement (see Figure 2).


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 correlation of 27 individual CpG methylation sites between pyrosequencing and Next Gen sequencing. Each plot displays one CpG site and the location is indicated at the top. The x-axis indicates Illumina sequencing, the y-axis pyrosequencing for percent methylation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The correlation of 27 individual CpG methylation sites between pyrosequencing and Next Gen sequencing. Each plot displays one CpG site and the location is indicated at the top. The x-axis indicates Illumina sequencing, the y-axis pyrosequencing for percent methylation.
Mentions: In total, 192.2 million reads 95 bp long were obtained from NG sequencing and 53.4 million (27.8%) possessed an average Phred score above 20 and were used for this analysis (Ewing and Green, 1998). 41.7 million reads (78.1%) were observed to contain one of the incorporated barcodes without mismatch and assigned to a corresponding sample for further analysis. Except one segment in the L2 gene, most segments included ~4–8 million reads (Figure S1A). For each sample, the read count varied from 2 to 6 million (Figure S1B). Most CpG sites (21/27) were covered by 0.6 to 1.8 million reads (Figure S1C), in total. Although three fragments did not amplify as robustly and had less reads (i.e., L1_2, L2_2, and L2_5 assays containing CpG sites 7034, 7091, 7136, 7145; 4427, 4437, 4441; and 5378, respectively), the correlation between CpG sites within these fragments between the PSQ and NGS assays had reasonable agreement (see Figure 2).

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