Genome Sequencing and Analysis of Tigecycline-Resistant Isolate

Total genomic DNA of the tigecycline-resistant isolate was extracted by Puregene Yeast/Bact Kit B (Qiagen, Maryland, US), and was sequenced by using Hiseq 4000 system (Illumina, San Diego, US) and PromethION platform (Nanopore, Oxford, UK). Hybrid assembly was performed by using Unicycler version 0.4.8 [28 (link)]. Antibiotic resistance genes were identified by ResFinder 3.2 [29 (link)] and CARD (https://card.mcmaster.ca/) with identity >80% and coverage >60%. Plasmid replicon typing was performed using PlasmidFinder v2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) with at least 95% identity and 60% coverage. Synteny analysis was performed using Easyﬁg [30 (link)]. Fragments >5 kb that were absent in at least one genome were detected by BLAST and were defined as genomic islands (GEIs) in this study as previously described [31 ]. Phylogenetic analysis with amino-acid sequences of Tet(X)s was performed by using the maximum likelihood method with default parameters by using Mega X Version 10.0.5 [32 (link)]. The amino acid sequences of Tet(X)s were submitted to ESPript 3 server [33 (link)] to perform the alignment and predict the secondary structure elements.

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Cheng Y., Chen Y., Liu Y., Guo Y., Zhou Y., Xiao T., Zhang S., Xu H., Chen Y., Shan T., Xiao Y, & Zhou K. (2020). Identification of novel tetracycline resistance gene tet(X14) and its co-occurrence with tet(X2) in a tigecycline-resistant and colistin-resistant Empedobacter stercoris. Emerging Microbes & Infections, 9(1), 1843-1852.

Publication 2020

Puregene Yeast/Bact Kit B Hiseq 4000 system PromethION platform

Amino acid sequences Amino acid sequences analysis Antibiotic resistance Genes Genome Genomic islands Hybrid Mega 10 Plasmid Replicon Synteny Tigecycline Yeast

Corresponding Organization : Southern University of Science and Technology

Other organizations : Zhejiang University, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences

Top 5 similar protocols

Variable analysis

independent variables

Total genomic DNA extraction method (Puregene Yeast/Bact Kit B)
DNA sequencing platforms (Illumina HiSeq 4000, Nanopore PromethION)
Hybrid assembly method (Unicycler version 0.4.8)
Antibiotic resistance gene identification (ResFinder 3.2, CARD)
Plasmid replicon typing (PlasmidFinder v2.1)
Synteny analysis (Easyﬁg)
Genomic island detection (BLAST)
Phylogenetic analysis of Tet(X) (MEGA X Version 10.0.5)
Secondary structure prediction of Tet(X) (ESPript 3 server)

dependent variables

Presence and characteristics of antibiotic resistance genes
Plasmid replicon types
Genomic island identification
Phylogenetic relationships of Tet(X)
Secondary structure of Tet(X)

control variables

Identity and coverage thresholds used for antibiotic resistance gene identification (>80% identity, >60% coverage)
Identity and coverage thresholds used for plasmid replicon typing (>95% identity, >60% coverage)

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