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Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Published on: February 24, 2015
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Multiplexed Reduced Representation Bisulfite Sequencing with Magnetic Bead Fragment Size Selection
William P Accomando1,2, Karin B Michels3
1Department of Epidemiology, Harvard School of Public Health, Boston, MA, 02115, USA. william_accomando@alumni.brown.edu.
Methods in Molecular Biology (Clifton, N.J.)
|December 11, 2017
Summary
This study introduces a magnetic bead-based method for targeted reduced representation bisulfite sequencing (RRBS). This approach enables efficient, multiplexed DNA methylation analysis of selected genomic regions across multiple samples.
Area of Science:
- Epigenetics and Genomics
- Molecular Biology Techniques
Background:
- Reduced representation bisulfite sequencing (RRBS) is crucial for genome-wide DNA methylation analysis in sequenced organisms.
- Existing RRBS methods require efficient selection of specific genomic contexts, particularly CpG-rich regions.
Purpose of the Study:
- To describe a novel magnetic bead-based approach for targeted selection in RRBS.
- To enable multiplexed RRBS analysis of up to 12 samples in a single sequencing lane.
Main Methods:
- Genomic DNA fragmentation using restriction endonucleases, followed by end-repair and A-tailing.
- Ligation of indexed, methylated adapters to DNA fragments.
- Magnetic bead-based selection of adapter-ligated fragments based on size to target specific genomic regions.
Main Results:
- Successful enrichment of desired DNA fragment sizes, enabling targeted sequencing.
- Demonstration of pooling up to 12 indexed libraries for multiplexed sequencing on an Illumina HiSeq2500.
- Comprehensive RRBS workflow including bisulfite treatment, PCR amplification, and final cleanup.
Conclusions:
- The described magnetic bead selection method offers precise control over targeted genomic regions for RRBS.
- Multiplexing capabilities significantly enhance the efficiency and throughput of DNA methylation studies.
