Clc genomic workbench v 10

For each Aconitum species, reads of the cp genome were assembled using CLC Genomic Workbench v10 (CLC Bio., Aarhus, Denmark). All the contigs were checked against the reference genome of A. chiisanense (KT820665), using BLAST (https://blast.ncbi.nlm.nih.gov/) and aligned contigs were oriented according to the reference genome. The complete cp genomes were then constructed using Geneious v4.8.5 (Biomatters Ltd., Auckland, New Zealand).
The annotation of cp genome sequence was performed using DOGMA (http://dogma.ccbb.utexas.edu/) [39 (link)] and start/stop codons and intron/exon boundaries were adjusted in Geneious v4.8.5. The tRNA was identified through tRNAscan-SE v2.0 [40 (link)]. The circular genome map was generated in OGDRAW (http://ogdraw.mpimp-golm.mpg.de/) [41 (link)].

The chloroplast genome was amplified in overlapping fragments according to the methods described by Yang et al. (2014) (link) at Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences. DNA samples were sheared into fragments of about 500 bp and used to construct libraries following the manufacturer’s instructions (Illumina, San Diego, CA, United States). Paired-end sequencing was conducted on Illumina HiSeq X-Ten platform. Raw reads were quality trimmed and the clean data were assembled after removing adapters using CLC Genomic Workbench v10 (CLC Bio., Aarhus, Denmark). Moreover, the raw sequencing data had been deposited in SRA (PRJNA504924). And then, the contigs were checked using BLAST searches² against the available complete chloroplast sequence of A. paniculata (NC_022451). Relative position and direction of each contigs were manually adjusted according to the reference genome. Finally, the complete chloroplast genome was acquired in Geneious v.8.1 (Kearse et al., 2012 (link)). Annotation of the chloroplast genome was conducted using OGDRAW (Lohse et al., 2013 (link)). The genome map of the species was illustrated with the help of CPGAVAS (Liu et al., 2012 (link)), and the annotated chloroplast genome sequences were submitted to NCBI under accession numbers: MF490441, MH045155, MH045156, and MH045157.

Genomic DNA for whole genome sequencing was prepared using MasterPure^™ DNA Purification Kit (Epicentre Technologies, Madison, Wisconsin). Pacific Biosciences sequencing was performed at the Science for Life laboratories sequencing platform at Uppsala University using Pacific Biosciences II technology and Illumina MiSeq was performed in-house using the NexteraXT technology. CLC Genomic Workbench v 10 (CLC Bio/Qiagen) with Microbial Genomics Module and Microbial Genome Finishing Tools was used for de novo assembly of reads and of reference assembly and variance analysis to K. pneumoniae LN824133. The sequence data including raw sequence reads and assembled contigs of the chromosome and plasmids have been deposited at NCBI with accession numbers CP024429-CP024436 under BioProject PRJNA348457.
The contigs were submitted to the ResFinder and PlasmidFinder databases at the Centre of Genomic Epidemiology (www.genomicepidemiology.org) to identify antibiotic resistance genes and plasmid replicons [26 (link)–28 (link)]. MLST was performed by submitting contigs at Institut Pasteur MLST Databases (http://www.pasteur.fr/mlst/).