Smrt analysis v2

Whole Genome Sequence analysis of mcr-1 containing isolates was performed to further characterize the E. coli strain including the plasmid carrying the mcr-1 gene and other genes associated with antimicrobial resistance [6 (link)]. The genome sequence of the mcr-1 containing isolate was determined using the Pacific Biosciences RSII system from DNA prepared by the Qiagen Genomic Tip 500/G kit (Qiagen, Hilden, Germany) following the manufacturer’s recommendations. De novo assembly was performed using SMRT®Analysis v2.3.0 (PacBio's bioinformatics software suite) with expected genome size of 5 Mbp and coverage of 30. The assembled sequence was analysed using Geneious software V8.0.5 (Biomatters, Auckland, New Zealand) and the online tools Resfinder, MLST, SeroTypeFinder and Plasmidfinder (http://genomicepidemiology.org/). The plasmid sequence was analysed in DNA plotter to generate a circular DNA map.

DNA of strain CFNEI73 was extracted according to standard protocols and sequenced by Macrogen (Seoul, South Korea). A 3 kilobase pair (kb)-library was prepared and run on an Illumina HiSeq sequencer to obtain 100 base pair (bp)-mated pair reads. A total of 10,599,614 paired reads were obtained, and 4,629,584 remained after trimming. A second sequencing protocol was done with PacBio at the Duke Center for Genomic and Computational Biology (Durham, NC, USA) with a 10 kb-library, obtaining 731,017,143 reads, filtered to 612,800,193. Sequences obtained were mixed with the Illumina reads to enhance the accuracy of the final assembly with genome coverage of 71×. Assembly was performed with SMRT Analysis v2.3.0 (Pacific Biosciences) and SPAdes v3.5.0 [16 (link)]. Annotation was conducted with RAST v4.0 [17 (link)], with manual curation. Strain CCGM7 was re-sequenced with PacBio at the Duke Center for Genomic and Computational Biology, with a 10 kb-library, obtaining 1,147,065,864 reads, filtered to 998,800,19. Reads were mixed with those obtained previously with Illumina [12 (link)], with genome coverage of 121×. Assembly and annotation were done as for strain CFNEI73.

The genomic DNA was extracted using Bacterial DNA Kit (Omega Bio-tek, Norcross, GA), according to the manufacturer’s instructions. The genome sequencing of S. pluranimalium TH11417 was performed on a PacBio RSII single-molecule real-time (SMRT) sequencing instrument (Pacific Biosciences, Menlo Park, CA). The average sequencing coverage was approximately 317× across the genome. The reads were assembled de novo using the hierarchical genome assembly process (HGAP) with the default settings of the SMRT Analysis v2.3.0 software package (Pacific Biosciences). The genome was annotated through the NCBI prokaryotic annotation pipeline (https://ncbi.nlm.nih.gov/).
The possible genomic islands (GIs) from TH11417 genome were predicted using IslandViewer 4 (http://www.pathogenomics.sfu.ca/islandviewer/), and prophage components were predicted according to the PHAST (http://phast.wishartlab.com/). Genome maps of TH11417 was generated using Circos v0.64 software [11 (link)]. The comparative analysis of prophage and type VII secretion system (T7SS) was generated using EasyFig v2.2 software (http://mjsull.github.io/Easyfig/files.html).

For the analysis of Iso-Seq data, we obtained high-quality reads of insert (ROI) by ConsensusTools script of SMRT Analysis v.2.3.0 software package from Pacific Biosciences. Then we used pbtranscript.py of SMRT Analysis v.2.3.0 software package to obtain full-length non-chimeric (FLNC) reads. For a small part of the chimeric ROI, they were marked as complete ROI by judging the 5′ primer, 3′ primer, presence of poly(A) and corresponding positional relationship. LSC 1.alpha (Au et al., 2012 (link)) was used to correct Iso-Seq. Finally, these corrected Iso-Seq reads were further used as an input file to identify AS events using PRAPI (Gao et al., 2018 (link)) with default parameters to identify the post-transcriptional regulation events including ATI, AS, and APA (Figure 2).

Rice mitochondria were isolated from callus by differential centrifugation method followed by continuous Percoll gradients as described by Heazlewood et al. [53 (link)]. The DNA was extracted from the rice mitochondria by CTAB and sequenced by NextOmics (Wuhan, China) on an Illumina HiSeq Xten machine with the 400 bp paired-end library, and on a PacBio RSII machine with 20 kb paired-end library.
The raw reads from PacBio RSII longer than 26 Kb were used as seed reads. The reads shorter than 26 Kb were corrected by the RS_PreAssembler.1 protocol with default settings from the Pacific Biosciences SMRT analysis (v2.3.0) software package. In addition, the raw reads from Illumina HiSeq Xten were also used to correct the genome by pilon (−-changes --vcf --fix bases --threads 5 --mindepth 10). Mitochondrial genomes were annotated using MITOFY [54 (link)]. The tRNA genes were searched using tRNAscan-SE [55 (link)]. Finally, the genome maps were drawn by OGDRAW [56 (link)].