Created on 19th December 2016
Advances in single molecule sequencing technology have enabled the investigation of the full catalogue of covalent DNA modifications. We present an assay, Modified DNA sequencing (MoD-seq), that leverages raw nanopore data processing, visualization and statistical testing to directly survey DNA modifications without the need for a large prior training dataset. We present case studies applying MoD-seq to identify three distinct marks, 4mC, 5mC, and 6mA, and demonstrate quantitative reproducibility across biological replicates processed in different labs. In a ground-truth dataset created via in vitro treatment of synthetic DNA with selected methylases, we show that modifications can be detected in a variety of distinct sequence contexts. We recapitulated known methylation patterns and frequencies in E. coli, and propose a pipeline for the comprehensive discovery of DNA modifications in a genome without a priori knowledge of their chemical identities.Show more
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