Vector nti program

Tetracycline resistant fosmid clones with different restriction patterns were sequenced at one end using the pCC1R primer (Epicentre) and the sequences compared as above. DNA from six representative fosmids was used to construct short insert genomic libraries of 0.5 kbp, which were then subjected to 91 bp paired-end sequencing using a HiSeq 2000 System sequencer (Illumina), providing a coverage of greater than 300-fold. Quality-filtered reads were assembled in contigs using Velvet software v.1.2.10². To close the sequence gaps, primers based on sequences at the extremes of the contigs were designed and used in PCR amplification. The amplicons were then sequenced. The Vector NTI program (Invitrogen) was used to assemble the contigs and PCR-derived sequences, and to predict putative open reading frames (ORFs). Predicted ORFs and deduced protein sequences were independently examined for homology against the non-redundant NCBI databases as described above.

The PC1 clones streaked on plates were used for screening by PCR and enzyme digestion and isolation of phagemids for sequencing. PCR was performed in a 10 μL reaction volume using a forward primer, CAPBSF1 (5′-ATGTGAGTTAGCTCACTCATTAGGC-3′), located upstream, and a reverse primer, GPIIICTR2 (5′-TGTCGTCTTTCCAGACGTTAGTAAATG-3′), located downstream of BglI cloning sites in the vector. The phagemid with any insert produced a fragment of 329 bp. The PCR product was checked by agarose gel electrophoresis. In the case of screening for the VLRB.HEL clones, the PCR product was digested with BglI and EcoRI to produce a small fragment (684 bp for VLRB.HEL.21) which allowed screening for VLRB.HEL.21 clones. The candidates were sequenced from DNA Sequencing Service of Operon, Fischer Scientific. Sequence contig assembly was done by Vector NTI program (Invitrogen) and alignment was done by ClustalW and ClustalX version 2 [27 (link)].

The NCBI database was used for the search of the CYP74-related genes. Primer construction and multiple sequence alignments were performed using the Vector NTI program (Invitrogen, USA). The BLAST analyses of the CYP74s were performed using the protein BLAST tool. The multiple alignments of selected CYP74 amino acid sequences and phylogenetic tree building were made with MEGA7 software [45 (link)]. Multiple alignment was performed using the Muscle method, the phylogenetic tree was built by means of the maximum likelihood method based on the Poisson correction model [46 ], and the bootstrap consensus tree was inferred from 1000 replicates [47 (link)]. The analysis involved 54 amino acid sequences.

Purified DNA from plasmids showing different restriction patterns were sent for sequencing to Eurofins Genomics (GATC Biotech, Constance, Germany). Individual libraries were constructed using the SPRIworks Fragment Library System I Kit (Beckman Coulter, Brea, CA, USA) and pair-end sequenced (2 × 150 bp runs) in a HiSeq sequencer (Illumina, San Diego, CA, USA). Quality-filtered reads, trimmed or non-trimmed depending on the plasmid, were de novo assembled in contigs using SPAdes v3.6.2 software [89 (link)], with a k value of 127 and employing the only-assembler settings. The plasmidSPAdes algorithm [90 (link)], which uses coverage as a parameter to remove chromosomal contigs, was also used for assembly. When plasmids were not merged into a single molecule after assembly, primers were designed based on the end of the contigs, used in PCR reactions, and the sequences of the amplicons employed to join the segments. The Vector NTI program (Invitrogen, Carlsbad, CA, USA) was used to align the sequences of contigs and PCR amplicons. The same program was used for open reading frame (ORF) prediction. Deduced protein sequences larger than 50 amino acids were compared to those in the NCBI’s non-redundant protein database and manually analyzed using BLASTp.

The bla_NDM-5-carrying plasmid pHNGD64-NDM was sequenced using PacBio platform and assembled using HGAP version 4.0. to analyze the genetic feature. BRIG software was used to comparative analysis with other plasmid sequences published in NCBI (Genbank accession number MT407547, MN915011, MN915010). The complete genome of plasmid was annotated and analysis using RAST (https://rast.nmpdr.org/rast.cgi, accessed on 21 September 2020), ISfinder (https://www-is.biotoul.fr/, accessed on 12 September 2020), Resfinder (https://cge.cbs.dtu.dk//services/ResFinder/, accessed on 12 September 2020) and Vector NTI program (Invitrogen).

PCR-based replicon typing (PBRT) was performed on all cfr/mcr-1-positive transconjugants/transformants using the primers as described previously [31 (link),32 (link)]. Plasmids pHNEP124 and pHNEP129 bearing the cfr and mcr-1 genes from transformants GDE6P124T and GDE6P129T were extracted and purified using a Qiagen plasmid midi kit (Qiagen, Hilden, Germany), and were subjected to sequencing by Illumina Miseq 2500 (Illumina, San Diego, CA, USA). The sequence reads were assembled into contigs using SOAP denovo version 2.04, and the gaps between the contigs were linked by PCR and sequencing. The complete sequences of plasmids pHNEP124 and pHNEP129 were analyzed and annotated by IS finder (https://www-is.biotoul.fr/), BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi), ResFinder (https://cge.cbs.dtu.dk//services/ResFinder/), RAST [33 (link)], and Vector NTI program (Invitrogen, San Diego, CA, USA).