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Figure 1. Direct evidence for the acquisition and demise of Tn6330 by DNA sequence alignment. (a) Comparison between ... Figure 1. Direct evidence for the acquisition and demise of Tn6330 by DNA sequence alignment. (a) Comparison between genetic structures of pHS30-1 and pKPC_CAV1374 demonstrates the possible translocation event mediated by Tn6330; the highlighted GT nucleotides represent the duplicated sequences surrounding IRL and IRR of Tn6330. (b) The circular form of ISApl1-mcr-1-orf detected by primers MCR1-R and MCR1-CD-F and sequencing. (c) The monomeric ISApl1 circular form detected by primers ISApl1-F2 and ISApl1-R. (d) Results of gel electrophoresis analysis of PCR products of different primer combinations; lane 1 matches the structure depicted in (b), lane 2 matches the structure shown in (c) and lane 3 matches the structures shown in (e), which are involved in generation of the 3679 bp circular form. All the primers are listed in Table S1. (f) Two nanopore single-molecule long-range reads depicting the demise of Tn6330, which occurs after losing the mcr-1-harbouring circular form. Both HS p_R1-RC (reverse complementary of read NB02:0.88:0.33|35–59:29138–29143) and HS p_R2-RC (reverse complementary of read NB02:0.88:0.29|32–54:22296–22304) reads were derived from nanopore raw reads of HS (populations collected after 30 passages). No reads were found to contain excised Tn6330 in sample HS30-1-C30, presumably due to low coverage of the sequencing data (Table S4). It should be noted that low identity between ONT reads and the reference plasmid may be attributed to the low accuracy of ONT sequencing reads. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model ( J Antimicrob Chemother, dkz117, The content of this slide may be subject to copyright: please see the slide notes for details.
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