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The BisPCR(2) method for targeted bisulfite sequencing.

Bernstein DL, Kameswaran V, Le Lay JE, Sheaffer KL, Kaestner KH - Epigenetics Chromatin (2015)

Bottom Line: DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification.We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR(2) compared to prior array-based approaches.This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104 USA.

ABSTRACT

Background: DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statistical significance, but are also high cost if performed genome-wide. Here, we describe a simplified targeted bisulfite sequencing approach in which DNA sequencing libraries are prepared following sodium bisulfite conversion and two rounds of PCR for target enrichment and sample barcoding, termed BisPCR(2).

Results: We have applied the BisPCR(2) technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR(2) compared to prior array-based approaches.

Conclusion: This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies.

No MeSH data available.


Related in: MedlinePlus

Validation of CpG loci differentially methylated in type 2 diabetic pancreatic islets by BisPCR2. Average percent CpG methylation in 5 non-diabetic and 5 type 2 diabetic human islet samples at loci previously shown to be differentially methylated in type 2 diabetic human islets, including aINS, bIRS1, cCDKN1A, and dPDE7B. Black arrows indicate CpGs analyzed previously by pyrosequencing by Dayeh and colleagues [18]. p value calculated by two-tailed t tests. *p < 0.05. Error bars indicate SEM.
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Fig4: Validation of CpG loci differentially methylated in type 2 diabetic pancreatic islets by BisPCR2. Average percent CpG methylation in 5 non-diabetic and 5 type 2 diabetic human islet samples at loci previously shown to be differentially methylated in type 2 diabetic human islets, including aINS, bIRS1, cCDKN1A, and dPDE7B. Black arrows indicate CpGs analyzed previously by pyrosequencing by Dayeh and colleagues [18]. p value calculated by two-tailed t tests. *p < 0.05. Error bars indicate SEM.

Mentions: We next sought to employ the BisPCR2 strategy to validate published differentially methylated loci in islets from type 2 diabetics [18]. We selected four genes, INS, IRS1, CDKN1A, and PDE7B, for validation. These loci were among those determined by Dayeh and colleagues to be differentially methylated in type 2 diabetic human islets in a genome-wide screen conducted with the Infinium HumanMethylation450 BeadChip array [18]. The insulin gene has also been described as differentially methylated in type 2 diabetic human islets through a candidate gene approach [19]. We designed PCR#1 primers targeting the region −112 to −336 base pairs upstream of the insulin transcription start site capturing four CpGs, three of which were previously reported to have increased DNA methylation in type 2 diabetic human islets [18]. We found all four CpGs measured had significantly increased DNA methylation (p < 0.05) with an average of 24% in non-diabetic compared to to 46% in type 2 diabetic samples (p < 0.0005) (Fig. 4a). This includes one CpG from the Infinium array that was not previously identified as differentially methylated, INS CpG #4 (cg24338752).Fig. 4


The BisPCR(2) method for targeted bisulfite sequencing.

Bernstein DL, Kameswaran V, Le Lay JE, Sheaffer KL, Kaestner KH - Epigenetics Chromatin (2015)

Validation of CpG loci differentially methylated in type 2 diabetic pancreatic islets by BisPCR2. Average percent CpG methylation in 5 non-diabetic and 5 type 2 diabetic human islet samples at loci previously shown to be differentially methylated in type 2 diabetic human islets, including aINS, bIRS1, cCDKN1A, and dPDE7B. Black arrows indicate CpGs analyzed previously by pyrosequencing by Dayeh and colleagues [18]. p value calculated by two-tailed t tests. *p < 0.05. Error bars indicate SEM.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4522100&req=5

Fig4: Validation of CpG loci differentially methylated in type 2 diabetic pancreatic islets by BisPCR2. Average percent CpG methylation in 5 non-diabetic and 5 type 2 diabetic human islet samples at loci previously shown to be differentially methylated in type 2 diabetic human islets, including aINS, bIRS1, cCDKN1A, and dPDE7B. Black arrows indicate CpGs analyzed previously by pyrosequencing by Dayeh and colleagues [18]. p value calculated by two-tailed t tests. *p < 0.05. Error bars indicate SEM.
Mentions: We next sought to employ the BisPCR2 strategy to validate published differentially methylated loci in islets from type 2 diabetics [18]. We selected four genes, INS, IRS1, CDKN1A, and PDE7B, for validation. These loci were among those determined by Dayeh and colleagues to be differentially methylated in type 2 diabetic human islets in a genome-wide screen conducted with the Infinium HumanMethylation450 BeadChip array [18]. The insulin gene has also been described as differentially methylated in type 2 diabetic human islets through a candidate gene approach [19]. We designed PCR#1 primers targeting the region −112 to −336 base pairs upstream of the insulin transcription start site capturing four CpGs, three of which were previously reported to have increased DNA methylation in type 2 diabetic human islets [18]. We found all four CpGs measured had significantly increased DNA methylation (p < 0.05) with an average of 24% in non-diabetic compared to to 46% in type 2 diabetic samples (p < 0.0005) (Fig. 4a). This includes one CpG from the Infinium array that was not previously identified as differentially methylated, INS CpG #4 (cg24338752).Fig. 4

Bottom Line: DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification.We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR(2) compared to prior array-based approaches.This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104 USA.

ABSTRACT

Background: DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statistical significance, but are also high cost if performed genome-wide. Here, we describe a simplified targeted bisulfite sequencing approach in which DNA sequencing libraries are prepared following sodium bisulfite conversion and two rounds of PCR for target enrichment and sample barcoding, termed BisPCR(2).

Results: We have applied the BisPCR(2) technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR(2) compared to prior array-based approaches.

Conclusion: This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies.

No MeSH data available.


Related in: MedlinePlus