Limits...
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 sequencing libraries. a Bioanalyzer gel visualizing the five amplicon fragments of a representative sample, ND1, following PCR#2. b Bioanalyzer electropherogram quantifying the amount of each fragment in ND1, illustrating the roughly equivalent amounts of all five amplicons. c Average reads per amplicon for each sample. ND non-diabetic, T2D type 2 diabetic.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: BisPCR2 DNA sequencing libraries. a Bioanalyzer gel visualizing the five amplicon fragments of a representative sample, ND1, following PCR#2. b Bioanalyzer electropherogram quantifying the amount of each fragment in ND1, illustrating the roughly equivalent amounts of all five amplicons. c Average reads per amplicon for each sample. ND non-diabetic, T2D type 2 diabetic.

Mentions: In this study, we selected five target loci for evaluation, as described below, and compared their DNA methylation profile in five non-diabetic and five type 2 diabetic human islet samples. Pancreatic islet donor information is provided in Table 1. Thus, for each of these ten biological samples, five PCR#1 amplicons were pooled, purified, and then used as template for the PCR#2 barcoding reaction. Target regions ranged in size from 171 to 298 bps (Table 2), and PCR#2 conditions were optimized to prevent amplification bias, particularly of smaller fragments, with the goal of balancing each library with roughly equivalent amounts of each amplicon (Fig. 2a, b).Table 1


The BisPCR(2) method for targeted bisulfite sequencing.

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

BisPCR2 DNA sequencing libraries. a Bioanalyzer gel visualizing the five amplicon fragments of a representative sample, ND1, following PCR#2. b Bioanalyzer electropherogram quantifying the amount of each fragment in ND1, illustrating the roughly equivalent amounts of all five amplicons. c Average reads per amplicon for each sample. ND non-diabetic, T2D type 2 diabetic.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: BisPCR2 DNA sequencing libraries. a Bioanalyzer gel visualizing the five amplicon fragments of a representative sample, ND1, following PCR#2. b Bioanalyzer electropherogram quantifying the amount of each fragment in ND1, illustrating the roughly equivalent amounts of all five amplicons. c Average reads per amplicon for each sample. ND non-diabetic, T2D type 2 diabetic.
Mentions: In this study, we selected five target loci for evaluation, as described below, and compared their DNA methylation profile in five non-diabetic and five type 2 diabetic human islet samples. Pancreatic islet donor information is provided in Table 1. Thus, for each of these ten biological samples, five PCR#1 amplicons were pooled, purified, and then used as template for the PCR#2 barcoding reaction. Target regions ranged in size from 171 to 298 bps (Table 2), and PCR#2 conditions were optimized to prevent amplification bias, particularly of smaller fragments, with the goal of balancing each library with roughly equivalent amounts of each amplicon (Fig. 2a, b).Table 1

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