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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

Schema of BisPCR2 method for targeted bisulfite sequencing. DNA sequencing libraries are prepared by bisulfite conversion of genomic DNA followed by two rounds of PCR for target enrichment (PCR#1) and subsequent sample barcoding (PCR#2). Partial adapter overhangs are added to target enrichment primers to permit simplified library preparation by PCR. PCR#1 amplicons are pooled prior to the PCR#2 reaction for each biological sample. Due to the presence of the unique barcodes, all PCR#2 amplicons can be pooled for a single next-generation sequencing run.
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Fig1: Schema of BisPCR2 method for targeted bisulfite sequencing. DNA sequencing libraries are prepared by bisulfite conversion of genomic DNA followed by two rounds of PCR for target enrichment (PCR#1) and subsequent sample barcoding (PCR#2). Partial adapter overhangs are added to target enrichment primers to permit simplified library preparation by PCR. PCR#1 amplicons are pooled prior to the PCR#2 reaction for each biological sample. Due to the presence of the unique barcodes, all PCR#2 amplicons can be pooled for a single next-generation sequencing run.

Mentions: Therefore, we have developed a novel approach for constructing targeted bisulfite NGS libraries that are prepared by bisulfite conversion of genomic DNA followed by two rounds of PCR, termed BisPCR2, eliminating the need for traditional DNA library preparation procedures (Fig. 1). In the BisPCR2 method, the entire library preparation process has been reduced to a single 50-min PCR reaction. We have validated the usefulness of this method in the context of type 2 diabetes, first confirming reported differences in DNA methylation at the imprinted MEG3 locus, and by validation of previous genome-wide findings of CpG risk loci identified in type 2 diabetic human islets [17, 18].Fig. 1


The BisPCR(2) method for targeted bisulfite sequencing.

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

Schema of BisPCR2 method for targeted bisulfite sequencing. DNA sequencing libraries are prepared by bisulfite conversion of genomic DNA followed by two rounds of PCR for target enrichment (PCR#1) and subsequent sample barcoding (PCR#2). Partial adapter overhangs are added to target enrichment primers to permit simplified library preparation by PCR. PCR#1 amplicons are pooled prior to the PCR#2 reaction for each biological sample. Due to the presence of the unique barcodes, all PCR#2 amplicons can be pooled for a single next-generation sequencing run.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Schema of BisPCR2 method for targeted bisulfite sequencing. DNA sequencing libraries are prepared by bisulfite conversion of genomic DNA followed by two rounds of PCR for target enrichment (PCR#1) and subsequent sample barcoding (PCR#2). Partial adapter overhangs are added to target enrichment primers to permit simplified library preparation by PCR. PCR#1 amplicons are pooled prior to the PCR#2 reaction for each biological sample. Due to the presence of the unique barcodes, all PCR#2 amplicons can be pooled for a single next-generation sequencing run.
Mentions: Therefore, we have developed a novel approach for constructing targeted bisulfite NGS libraries that are prepared by bisulfite conversion of genomic DNA followed by two rounds of PCR, termed BisPCR2, eliminating the need for traditional DNA library preparation procedures (Fig. 1). In the BisPCR2 method, the entire library preparation process has been reduced to a single 50-min PCR reaction. We have validated the usefulness of this method in the context of type 2 diabetes, first confirming reported differences in DNA methylation at the imprinted MEG3 locus, and by validation of previous genome-wide findings of CpG risk loci identified in type 2 diabetic human islets [17, 18].Fig. 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