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. 2016 Jan 29;44(2):730-43.
doi: 10.1093/nar/gkv1498. Epub 2015 Dec 23.

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology

Lingxiang Zhu  1 , Jun Zhong  2 , Xinmiao Jia  3 , Guan Liu  4 , Yu Kang  2 , Mengxing Dong  3 , Xiuli Zhang  3 , Qian Li  3 , Liya Yue  3 , Cuidan Li  3 , Jing Fu  3 , Jingfa Xiao  2 , Jiangwei Yan  2 , Bing Zhang  5 , Meng Lei  5 , Suting Chen  4 , Lingna Lv  4 , Baoli Zhu  6 , Hairong Huang  7 , Fei Chen  8
Affiliations

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology

Lingxiang Zhu et al. Nucleic Acids Res. .

Abstract

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBC's genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three (m6)A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring 'the methylated-motif-site ratio' and 'the methylated-read ratio', we explored the methylation status of each modified site and sequence-read to obtain the 'precision methylome' of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.

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Figures

Figure 1.
Figure 1.
Phylogenetic analysis of Mycobacterium tuberculosis complex (MTBC) strains. Blue indicates that strains belong to lineage 4 (L4, Euro-American lineage). Pink indicates that strains belong to L3 (East African-Indian lineage). Red indicates that strains belong to L2 (East Asian lineage). Orange indicates that strains belong to L1 (Indo-Oceanic lineage). Purple indicates that strains belong to L5 and L6 (West African 1 and West African 2 lineage). Dark blue indicates that strains belong to L7 (Ethiopia lineage). Green indicates that strains belong to animal-adapted lineage (L8). The strains in red boxes represent the 12 MTBC genomes we finished here.
Figure 2.
Figure 2.
Distribution of the methylated read ratio of three motifs in the MTBC stains. The horizontal axis shows the strain name. The vertical axis shows the number of motifs with diverse methylated read ratio (30–60%, 60–90%, 90–100%). The methylated read ratio indicates the percentage between the reads containing the methylated base and the total reads mapped to the site. For example, as for one methylated site, if its methylated read ratio is 60% and there are 100 reads covering (mapped to) this methylated site, this means that 60 reads contain the methylated base and the other 40 reads have no methylation. (A) Distribution of the methylated read ratio of CTCCAG motif in the MTBC stains. (B) Distribution of the methylated read ratio of GATN4RTAC motif in the MTBC stains. (C) Distribution of the methylated read ratio of CACGCAG motif in the MTBC stains.
Figure 3.
Figure 3.
Three MTase genes and corresponding methylated sequence motifs in 12 MTBC strains. M. bovis 30 strain is located in the central position and the 11 other MTBC strains are located around it. Only active MTase genes were shown in the figure. The red circles mark the order change of the two MTase genes due to a large-scale inversion (about 1.8 Mbp) in Mtb 2242.
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