3.7 DNA modification analysis
DNA modifications were determined in the mt genome of O.sinensis in parallel with the acquisition of primary sequence data by SMRT sequencing，based on analysis of the kinetics of DNA synthesis reactions.DNA modifications differ by various modification types，including 5-methylcytosine(5mC)，6-methyladenine(6mA)，4-methylcytosine(4mC)and 5-hydroxymethylcytosine(5-hmC)，which can be identified by a series of methyltransferases at specific motifs.A genomic position is covered by several sequenced DNA fragments，and the modQV score shows the consistency by which a specific modification is observed.In the O.sinensis mt genome，a total of 1604 sites were determined with an average modQV score of 24.68 at an average coverage of approximately 96x(Fig.4).There were 783 modification sites located on the forward strand versus 821 located on the backward strand(Fig.4).The plot of modification scores against sequencing coverage displayed a dominant signal for modified adenosines and thymine bases(pink and burgundy dots in Fig.4).Twenty-eight 4mC(0.13%)and ten 6m A(0.017%)modification sites were identified in the O.sinensis mt genome(Fig.1，Table 6).The 6mA levels were lower than previously reported for nucleotide methylation(0.048%～0.21%)in eukaryotic nuclear genomes(Mondo et al.，2017).Most 6mA and 4mC were distributed in intergenic regions(between tRNA and nad4L/nad6/cox2，or between tRNA and tRNA)or in intron regions of different genes(eg.nad1—nad2，cox1-cox2，cob，rnl)，with only three located in the genes.Two 4mC were methylated on the backward strand in the regions of nad2 and nad5 genes，and one 6mA was methylated in the nad4L region(Table 6).The hypermethylation in promoters and introns，and hypomethylation in exons，are also found in the nuclear genome of eukaryotes(Volpe，2005).It is reported that methylation is strand-specific within the mt genome(Bellizzi et al.，2013).However，methyl modifications in the O.sinensis mt genome were observed on both strands，including 4mCand 6mA modifications(Table 6).
Fig.4 DNA modifications in the O.sinensis mt genome
At each genomic position，modification quality value(modQV)were computed as the—10 log(p-value)for representing a modified base position，based on the distributions of the kinetics of interpulse durations(IPD ratios)from all reads covering this position and from in silico kinetic reference values.Each dot represents a position on either strand with a modQV larger than 20.The color specified the nucleotide base，on which the modification was detected.Adenosines were colored in red，guanosines were colored in green，cytimidines were colored in blue，and thymines were colored in purple.
Table 6 6mA and 4mC in the mt genome of O.sinensis(https://www.daowen.com)
Continued table
Because DNA methylation is diverse，widespread，and often deposited by a diverse set of methyltransferases at specific target sequences(motif)，a comprehensive understanding of the distribution and diversity of methylation motifs will benefit comprehension of methylation function and evolutionary history of the mt genome.Eight modification motifs“AANNNm4CAGCANNANNNNA”，five of which were recognized by 4mC methyltransferases，were detected in the O.sinensis genome by SMRT sequencing with a mean QV of 44.4 at a mean coverage of 90.4x.Among the five methylation sites，four were in the introns of PCGs(cob，cox1，and rnl)，one was in the an intergenic region(tRNA_I_tRNA_G).