Sparse Sequencing permits accurate and efficient quantification of genome-wide cytosine modification levels
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5-methylcytosine (5mC) and 5-hydroxymethycytosine (5hmC) play crucial roles in epigenetic gene regulation, with dynamic levels in development and disease. Current methods for quantifying 5mC and 5hmC levels genome-wide are typically limited in either throughput or accuracy. Using computational down-sampling of deeply-sequenced data sets, we demonstrate that sparse base-resolution sampling (<0.24% of the mouse genome) can precisely measure mammalian genomic 5mC and 5hmC levels (error <5%), even when levels are low (<0.3%). We then demonstrate that Sparse Sequencing (Sparse-Seq) using chemical and enzymatic methods shows high accuracy and less variability than mass spectrometry-based methods in global quantification. The added ability of Sparse-Seq to dissect sequence context permitted resolution of distinct time-courses for the emergence of 5mCpH versus 5hmCpG in developing brains. Sparse-Seq offers a highly accessible and broadly useful approach for quantifying both 5mC and 5hmC levels, allowing for economical and high-throughput analysis of epigenomic dynamics.
