Lasergene version 8

Sequencing of Rickettsia-positive nPCR amplicons was conducted by Macrogen Inc. (Daejeon, Korea). The obtained sequences were compared for similarity to sequences deposited in GenBank using BLAST. Gene sequences, excluding the primer regions, were aligned using the multisequence alignment program in Lasergene version 8 (DNASTAR, USA), and phylogenetic analysis performed using MEGA 6 software.
Phylogenetic trees were constructed in CLUSTAL W of the MegAlign Program (DNASTAR, USA) based on the alignment of rickettsial gene sequences obtained following nPCR using the neighbor-joining method and bootstrap analysis (1,000 reiterations) carried out according to the Kimura 2-parameter method. Pairwise alignments were performed with an open-gap penalty of 10 and a gap extension penalty of 0.5. All positions containing alignment gaps and missing data were eliminated during the pairwise sequence comparison (pairwise deletion).

Sequencing of PCR amplicons from rodent-borne pathogens was conducted by Solgent Inc. (Daejeon, Korea). The obtained sequences were compared for similarity with the sequences deposited in the GenBank using BLAST. The gene sequences excluding the primer regions were aligned using the multisequence alignment program of Lasergene version 8 (DNASTAR, USA), and phylogenetic analysis was performed using the MEGA 6 software.
Phylogenetic trees were constructed using ClustalW of the MegAlign Program (DNASTAR, USA) based on the alignments of positive gene sequences using the neighbour-joining method. Bootstrap analysis (1,000 replicates) was performed according to the Kimura 2-parameter method. Pairwise alignments were performed with an open-gap penalty of 10 and a gap extension penalty of 0.5.

The amplified PCR products were purified using QIAquick PCR purification kits (QIAGEN, Hilden, Germany) and sequenced using PCR primers at Solgent Inc. (Daejeon, Korea). The sequences obtained in this study were compared to GenBank sequences using BLAST. Gene sequences, excluding the primer regions, were aligned using the multisequence alignment program in Lasergene version 8 (DNASTAR, USA). The nucleotide sequences obtained from the PCR amplifications performed in this study were registered and assigned the following GenBank accession numbers: Tick1 (MW481245) and Tick29 (MW481246) for the ankA gene; Tick29 (MW481247), Tick2 (MW481248), Tick17-1 (MW481249), Tick17-2 (MW481250), Tick15 (MW481251), Tick5 (MW481252), Tick6 (MW481253), Tick7 (MW481254), Tick18 (MW481255), Tick30 (MW481256), Tick19 (MW481257), Tick21 (MW481258), and Tick24 (MW481259) for the gltA gene; Tick25 (MW475155), Tick12 (MW475156), and Tick19 (MW475157) for the 18S rDNA.
Phylogenetic trees were constructed using ClustalW of the MegAlign Program (DNASTAR, USA) based on the alignments of positive gene sequences using the neighbor-joining method. Bootstrap analysis (1,000 replicates) was performed according to the Kimura 2-parameter method. Pairwise alignments were performed with an open-gap penalty of 10 and a gap extension penalty of 0.5.