TY - JOUR
T1 - Focused, high accuracy 5-methylcytosine quantitation with base resolution by benchtop next-generation sequencing
AU - Masser, Dustin R.
AU - Berg, Arthur
AU - Freeman, Willard M.
N1 - Funding Information:
The authors thank David Stanford in the Molecular Genetics Core Facility at the Penn State Hershey College of Medicine for Sanger sequencing assistance, Georgina Bixler in the Genome Sciences Facility at the Penn State Hershey College of Medicine for quantitative PCR assistance, Anna Salzberg for establishing our ESME workflow, Colleen Van Kirk for mouse retina and cerebellum DNA, and Sean Boswell for figure generation. This work was supported by NIH grants DA029405, EY021716, and AG026607 and Commonwealth of Pennsylvania, Department of Health, Commonwealth Universal Research Enhancements SAP 4100055576.
PY - 2013
Y1 - 2013
N2 - Background: The growing interest in the role of epigenetic modifications in human health and disease has led to the development of next-generation sequencing methods for whole genome analysis of DNA methylation patterns. However, many projects require targeted methylation analysis of specific genes or genomic regions. We have developed an approach, termed BiSulfite Amplicon Sequencing (BSAS), for hypothesis driven and focused absolute DNA methylation analysis. This approach is applicable both to targeted DNA methylation studies as well as to confirmation of genome-wide studies. Results: BSAS uses PCR enrichment of targeted regions from bisulfite-converted DNA and transposome-mediated library construction for rapid generation of sequencing libraries from low (1 ng) sample input. Libraries are sequenced using the Illumina MiSeq benchtop sequencer. Generating high levels of sequencing depth (>1,000 ×) provides for quantitatively precise and accurate assessment of DNA methylation levels with base specificity. Dual indexing of sequencing libraries allows for simultaneous analysis of up to 96 samples. We demonstrate the superior quantitative accuracy of this approach as compared to existing Sanger sequencing methods. Conclusions: BSAS can be applied to any genomic region from any DNA source, including tissue and cell culture. Thus, BSAS provides a new validation approach for rapid and highly quantitative absolute CpG methylation analysis of any targeted genomic regions in a high throughput manner.
AB - Background: The growing interest in the role of epigenetic modifications in human health and disease has led to the development of next-generation sequencing methods for whole genome analysis of DNA methylation patterns. However, many projects require targeted methylation analysis of specific genes or genomic regions. We have developed an approach, termed BiSulfite Amplicon Sequencing (BSAS), for hypothesis driven and focused absolute DNA methylation analysis. This approach is applicable both to targeted DNA methylation studies as well as to confirmation of genome-wide studies. Results: BSAS uses PCR enrichment of targeted regions from bisulfite-converted DNA and transposome-mediated library construction for rapid generation of sequencing libraries from low (1 ng) sample input. Libraries are sequenced using the Illumina MiSeq benchtop sequencer. Generating high levels of sequencing depth (>1,000 ×) provides for quantitatively precise and accurate assessment of DNA methylation levels with base specificity. Dual indexing of sequencing libraries allows for simultaneous analysis of up to 96 samples. We demonstrate the superior quantitative accuracy of this approach as compared to existing Sanger sequencing methods. Conclusions: BSAS can be applied to any genomic region from any DNA source, including tissue and cell culture. Thus, BSAS provides a new validation approach for rapid and highly quantitative absolute CpG methylation analysis of any targeted genomic regions in a high throughput manner.
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U2 - 10.1186/1756-8935-6-33
DO - 10.1186/1756-8935-6-33
M3 - Article
AN - SCOPUS:84885368581
SN - 1756-8935
VL - 6
JO - Epigenetics and Chromatin
JF - Epigenetics and Chromatin
IS - 1
M1 - 33
ER -