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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

BisPCR2 DNA methylation analysis confirms increased CpG methylation in type 2 diabetic human islets at the MEG3 locus. a Average percent CpG methylation at the MEG3 locus for five non-diabetic and five type 2 diabetic human islet samples measured by BisPCR2. p values calculated by a two-tailed t test. *p < 0.05. Error bars indicate SEM. b Quantification of average percent CpG methylation by pyrosequencing using the same samples and same MEG3 PCR primer sequences as in a. Only 2 of 19 CpGs are covered in the pyrosequencing assay. Data displayed as in a.
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Fig3: BisPCR2 DNA methylation analysis confirms increased CpG methylation in type 2 diabetic human islets at the MEG3 locus. a Average percent CpG methylation at the MEG3 locus for five non-diabetic and five type 2 diabetic human islet samples measured by BisPCR2. p values calculated by a two-tailed t test. *p < 0.05. Error bars indicate SEM. b Quantification of average percent CpG methylation by pyrosequencing using the same samples and same MEG3 PCR primer sequences as in a. Only 2 of 19 CpGs are covered in the pyrosequencing assay. Data displayed as in a.

Mentions: Target enrichment primers were designed to amplify a 298 base pair region within the MEG3 promoter at position −188 to −493 relative to the transcription start site. This amplicon covered 19 CpGs, and the average CpG methylation across the region was significantly increased from 43% in non-diabetic to 61% in type 2 diabetic human islets (p < 0.0001), confirming the report by Kameswaran and colleagues. Of the 19 CpGs covered, 14 had significantly increased CpG methylation in type 2 diabetics (p < 0.05) (Fig. 3a). To further corroborate our findings, using primers directed to the same target region, we technically validated our results with pyrosequencing. Although the same target region was analyzed, the fluorescence-based pyrosequencing reaction covered only 2 of the 19 CpGs within the amplicon, #15 and #16. These 2 CpGs showed comparable levels of CpG methylation in the non-diabetic and type 2 diabetic samples as the BisPCR2 method (Fig. 3b). Thus, we were able to technically validate our results with pyrosequencing, and analyze ten times as many CpGs with the BisPCR2 method.Fig. 3


The BisPCR(2) method for targeted bisulfite sequencing.

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

BisPCR2 DNA methylation analysis confirms increased CpG methylation in type 2 diabetic human islets at the MEG3 locus. a Average percent CpG methylation at the MEG3 locus for five non-diabetic and five type 2 diabetic human islet samples measured by BisPCR2. p values calculated by a two-tailed t test. *p < 0.05. Error bars indicate SEM. b Quantification of average percent CpG methylation by pyrosequencing using the same samples and same MEG3 PCR primer sequences as in a. Only 2 of 19 CpGs are covered in the pyrosequencing assay. Data displayed as in a.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: BisPCR2 DNA methylation analysis confirms increased CpG methylation in type 2 diabetic human islets at the MEG3 locus. a Average percent CpG methylation at the MEG3 locus for five non-diabetic and five type 2 diabetic human islet samples measured by BisPCR2. p values calculated by a two-tailed t test. *p < 0.05. Error bars indicate SEM. b Quantification of average percent CpG methylation by pyrosequencing using the same samples and same MEG3 PCR primer sequences as in a. Only 2 of 19 CpGs are covered in the pyrosequencing assay. Data displayed as in a.
Mentions: Target enrichment primers were designed to amplify a 298 base pair region within the MEG3 promoter at position −188 to −493 relative to the transcription start site. This amplicon covered 19 CpGs, and the average CpG methylation across the region was significantly increased from 43% in non-diabetic to 61% in type 2 diabetic human islets (p < 0.0001), confirming the report by Kameswaran and colleagues. Of the 19 CpGs covered, 14 had significantly increased CpG methylation in type 2 diabetics (p < 0.05) (Fig. 3a). To further corroborate our findings, using primers directed to the same target region, we technically validated our results with pyrosequencing. Although the same target region was analyzed, the fluorescence-based pyrosequencing reaction covered only 2 of the 19 CpGs within the amplicon, #15 and #16. These 2 CpGs showed comparable levels of CpG methylation in the non-diabetic and type 2 diabetic samples as the BisPCR2 method (Fig. 3b). Thus, we were able to technically validate our results with pyrosequencing, and analyze ten times as many CpGs with the BisPCR2 method.Fig. 3

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