Methanococcus aeolicus Genome Sequencing

Genomic DNA from Methanococcus aeolicus PL15/H^p was purified using a Monarch Genome Purification kit (T3010, NEB, Ipswich, MA, USA) and the DNA sample was sheared to an average size of ∼ 10 kb using the G-tube protocol (Covaris, Woburn, MA, USA). DNA libraries were prepared using a SMRTbell express template prep kit 2.0 (100–938–900, Pacific Biosciences, Menlo Park, CA, USA) and ligated with hairpin barcoded adapter lbc1-lbc1. Incompletely formed SMRTbell templates were removed by digestion with a combination of exonuclease III and exonuclease VII (NEB, Ipswich, MA, USA). The qualification and quantification of the SMRTbell libraries were made on a Qubit fluorimeter (Invitrogen, OR) and a 2,100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). SMRT sequencing was performed using an SQ1 (Pacific Biosciences, Menlo Park, CA, USA) based on the multiplex protocol for 10 kb SMRTbell library inserts. Sequencing reads were collected and de novo assembled using the Microbial Assembly version 10.1.0.1119588 program with default quality and read length parameters. In addition to genome assembly (Chin et al., 2013 (link)), the SMRT Analysis pipeline from Pacific Biosciences¹ enables the determination of the epigenetic status of sequenced DNA by identifying the m6A and m4C modified motifs (Flusberg et al., 2010 (link); Clark et al., 2012 (link); Korlach and Turner, 2012 (link)).