Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing

5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are modified versions of cytosine in DNA with roles in regulating gene expression. Using whole genomic DNA from mouse cerebellum, we have benchmarked 5mC and 5hmC detection by Oxford Nanopore Technologies sequencing against other standard techniques. In addition, we assessed the ability of duplex base-calling to study strand asymmetric modification. Nanopore detection of 5mC and 5hmC is accurate relative to compared techniques and opens new means of studying these modifications. Strand asymmetric modification is widespread across the genome but reduced at imprinting control regions and CTCF binding sites in mouse cerebellum. This study demonstrates the unique ability of nanopore sequencing to improve the resolution and detail of cytosine modification mapping. Cerebellum tissues were obtained from two 8-week-old female C57BL/6J mice, from which high molecular weight genomic DNA was extracted using the QIAGEN MagAttract HMW DNA Kit (QIAGEN, 67563). Cerebellar gDNA was sheared using a Covaris g-TUBE (Covaris, 520079) to a target length of 8 kb. Whole genome sequencing (WGS) libraries were prepared in duplicate for each biological replicate, using the Ligation Sequencing Kit V14 standard protocol (Oxford Nanopore Technologies, SQK-LSK114) with reagents from the NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (New England Biolabs, E7180S). 150 ng of each sample was loaded onto PromethION (Oxford Nanopore Technologies, PRO-SEQ048) flow cells (Oxford Nanopore Technologies, FLO-PRO114HD). All sequencing runs were manually stopped after 72 hours.