MicrobeMod:一个用于鉴定原核甲基化和纳米孔测序限制性修饰的计算工具包

Alexander Crits-Christoph, Shinyoung Clair Kang, Henry H Lee, Nili Ostrov
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摘要

细菌和古细菌利用限制性修饰(R-M)系统通过不同序列特异性的DNA甲基转移酶甲基化其基因组来区分自身和外源DNA,这些免疫系统通常在菌株水平上有所不同。鉴定活性甲基化模式和R-M系统可以揭示重组DNA引入或噬菌体感染的障碍。在这里,我们提出了计算MicrobeMod工具包,用于鉴定细菌基因组中的5mC和6mA甲基化序列基序和R-M系统,使用天然DNA的纳米孔测序。我们对一组具有已知特异性表达甲基转移酶的参考大肠杆菌菌株进行了基准测试。然后,我们将这些分析应用于31种不同的细菌和古细菌生物体,以揭示以前未探索的表观遗传学菌株的甲基化模式,发现原核5-甲基胞嘧啶可能比以前报道的更常见。总之,MicrobeMod可以在测序深度低至10倍的情况下快速揭示原核基因组中新的表观遗传学,并且只需要天然DNA。该工具包可用于推进细菌甲基化的基础知识和指导策略,以克服非模式微生物遗传易感性的R-M障碍。
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MicrobeMod: A computational toolkit for identifying prokaryotic methylation and restriction-modification with nanopore sequencing
Bacteria and archaea use restriction-modification (R-M) systems to distinguish self from foreign DNA by methylating their genomes with DNA methyltransferases with diverse sequence specificities, and these immunity systems often vary at the strain level. Identifying active methylation patterns and R-M systems can reveal barriers to the introduction of recombinant DNA or phage infection. Here, we present the computational MicrobeMod toolkit for identifying 5mC and 6mA methylation sequence motifs and R-M systems in bacterial genomes using nanopore sequencing of native DNA. We benchmark this approach on a set of reference E. coli strains expressing methyltransferases with known specificities. We then applied these analyses to 31 diverse bacterial and archaeal organisms to reveal the methylation patterns of strains with previously unexplored epigenetics, finding that prokaryotic 5-methylcytosine may be more common than previously reported. In summary, MicrobeMod can rapidly reveal new epigenetics within a prokaryotic genome sequenced with Oxford Nanopore R10.4.1 flow cells at sequencing depths as low as 10x and only requires native DNA. This toolkit can be used to advance fundamental knowledge of bacterial methylation and guide strategies to overcome R-M barriers of genetic tractability in non-model microbes.
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