Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing

1] Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota, USA. [2].
Nature Biotechnology (Impact Factor: 39.08). 11/2012; 31(6). DOI: 10.1038/nbt.2432
Source: PubMed

ABSTRACT Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

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    • " acquisitions of for - eign genetic elements . Functional studies of SPI - 5 with and without these GIs might be important to examine the possible role of both SPI5 - GIs . Both SPI5 - GIs contained genes en - coding a methylase , which could potentially regulate chro - mosome replication , cell cycle events , pathogenicity , and gene expression ( Fang et al . , 2012 ; Davis et al . , 2013 ) . The SPI - 5 genes could be considered targets for re - sequencing and biomarkers to rapidly differentiate lineages II and III . We performed positive selection tests for pipA and pipB using codon - based Z tests in MEGA6 , indicating that these two genes were under positive selection . Positive se - lection pl"
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    • "The Pacific Biosciences (PacBio) RS can yield average sequence reads of greater than 2500 bp; however, to maximize accuracy, circular consensus sequencing (CCS) of shorter fragments (b1500 bp) provides multiple passes of the fragment and decreases sequencing errors (Au et al., 2012). Previous studies utilizing the PacBio RS SMRT chip have sequenced genomes (Thudi et al., 2012; Zhang et al., 2012), expressed antibody transcripts (Larsen and Smith, 2012), DNA methylation (Clark et al., 2012; Fang et al., 2012; Lluch-Senar et al., 2013; Murray et al., 2012; Zillner and Nemeth, 2012), kinetic variation in base modifications (Schadt et al., 2013) and electrosynthetic microbiomes (Fichot and Norman, 2013). To the best of our knowledge, no studies have examined the efficacy of this platform on 16S rRNA amplicons from environmental samples. "
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