SMRT Bell Library Construction and SMRT Sequencing

Bacterial genomic DNA (gDNA) was purified from B. subtilis T30 cells using the gDNA purification kit purchased from Qiagen. The genome was sequenced using the Pacific Biosciences (PacBio) RSII sequencing platform as previously described (Flusberg et al., 2010; Clark et al., 2012). Briefly, SMRT bell libraries were constructed from a gDNA sample sheared to ∼10–20 kb using the G-tubes protocol (Covaris). The sheared ends were repaired and ligated to PacBio hairpin adapters according to the manufacturer’s protocol. Incompletely formed SMRT bell templates and linear DNA were digested by Exonucleases III and VII (NEB). DNA quantification and the quality of the library was analyzed using the Qubit fluorimeter (Invitrogen) and 2100 Bioanalyzer (Agilent Technology). Two 18-kb SMRT bell libraries were prepared according to PacBio sample preparation protocols for 20 kb libraries and sequenced using C2-P4 chemistry (4 SMRT cells, 120 min collection times). To enhance interpulse duration (IPD) ratios signal, m5C modified bases in the genomic DNA were converted to hm5C by treatment with Tet2 enzyme (catalytic domain, NEB). Tet2 oxidation was carried out according to the protocol for EM-seq kit (NEB, E7120S) (Shen et al., 2018). SMRT library DNA (1 μg) was incubated with Tet2 enzyme in a reaction buffer (50 mM HEPES, pH 8.0, 100 μM Fe(NH₄)₂(SO4)₂, 2 mM ascorbate, 1 mM α-ketoglutarate, 1 mM ATP, 1 mM TCEP) at 37°C for 1 h. Reaction was terminated by addition of stop solution provided in the kit. Software provided by Pacific Biosciences or developed in house was used to detect the modified bases (IPD ratios). An IPD ratio > 1 means that the sequencing DNA polymerase slowed down (relative to the control) at this modified nucleotide position.

SMRT sequencing of E. coli genomic DNA modified with M.BisIII expression: BisIIIM gene (PCR fragment) was inserted into a T7 expression vector and transferred into E. coli T7 Express (a Dam-deficient strain) by transformation. E. coli genomic DNA was extracted from overnight cell culture (IPTG-induced) and the gDNA was treated with Tet2 catalytic domain enzyme. The Tet2-treated DNA was used for SMRT sequencing and for detection of CCWGG modified sites based in the IPD ratio.

Modified deoxy-oligoribonucleotide (oligos) substrates were synthesized by SibEnzyme Ltd. (Russia). The sequence and modified base composition of the oligos are shown in Table 1. Duplex DNA oligos were prepared by annealing two complementary oligos which differ from each other by the presence or absence of m5C within the recognition sequence GCNGC and used in BisI digestions.

Modified DNA oligo sequences used in this study.