Computational protocol: Isolation and Characterization of Two Novel Plasmids from Pathogenic Leptospira interrogans Serogroup Canicola Serovar Canicola Strain Gui44

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Protocol publication

[…] As mentioned above, total genomic DNA of strain Gui44 including both chromosome and plasmid DNA was sequenced with Hiseq 2000 system. Sequenced reads were aligned to the reference genome (including the two chromosomes and two plasmids) of the strain Gui44 by Bowtie2 using the default parameters . When the reads were exclusively mapped to the simple location of the genome, they were counted by using the idxstats module in SAMtools software . The relative copy number was calculated by: aligned reads numbers× average read length/(corresponding chromosome or plasmid size), as well as by qPCR on an ABI 7500 fast real-time PCR system (Applied Biosystems of Thermo Fisher Scientific Inc., Waltham, MA, USA) with a FastStart Universal SYBER Green Master mix (Roche Diagnostics., Indianapolis, USA) according to manufacturer's instructions. PCR primers were designed using Beacon Designer 7 software (http://www.premierbiosoft.com/molecular_beacons/). Eight-fold serial dilutions of the plasmids from Peasy-T1 (Beijing Transgen Biotech Co., LTD, Beijing, China) and template DNA were used to construct standard curves of all plasmids. Reactions were performed in duplicate in a total reaction volume of 20 µL. A 92 bp fragment of a replication initiation factor (dnaA), a single-copy gene in the larger chromosome, was used as a reference gene. A 94 bp fragment designated P1 that is unique to pGui1 and a 100 bp fragment designated P2 which is specific to pGui2 were amplified (all primers used for qPCR are listed in ). The reactions were performed under the following conditions: 10 min at 95°C, followed by 40 cycles of 30 sec at 95°C, 30 sec at 56°C, and 30 sec at 72°C. The experiment was carried out in triplicate and averages were reported. The relative copy number of plasmids was calculated using Nrelatives = (1+E)−ΔC T, where E stands for the PCR amplification efficiency and ΔCT was for the difference in threshold cycle number between the reference gene and target gene. […]

Pipeline specifications

Software tools Bowtie2, SAMtools, Beacon Designer
Application qPCR
Organisms Leptospira interrogans serovar Canicola, Leptospira interrogans