Editseq program

The positive PCR amplicons of ten randomly selected samples obtained from domestic and wild goats were further sequenced to gain information about the individual N gene structure. PCR products were purified using QIAquick PCR purification kit (Qiagen, GmbH) according to the manufacturer’s instructions. Quality and concentration of purified PCR products were confirmed as mentioned in the above section. The purified PCR products were sequenced at Seqlab-Sequence Laboratories GmbH (Göttingen, Germany), and the sequences were analyzed using FinchTV (version, 1.4.0). For confirming sequence identity, sequence data was subjected to Nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the global database. Additionally, all sequences were further analyzed and compared by Clustal-V and Clustal-W pairwise multiple sequence alignment using MegAlign (DNASTAR Inc., Madison, WI, USA). The phylogenetic tree was constructed where bootstrapping was performed by creating 1000 trials. Similarly, the nucleotide sequences were converted to amino acid sequences using the translate DNA step of the EditSeq program (DNASTAR Inc.) and the phylogenetic tree was constructed using the MegAlign program.

The AT-skew = (A − T)/(A + T) and the GC-skew = (G − C)/(G + C) were computed for the α strand of the Brachyuran mtDNAs in order to evaluate the compositional biases^{53 (link)}. The base compositions were determined with the EditSeq program from the Lasergene software package (DNAStar, Madison, WI).
The total number of codons present in the mitochondrial protein-coding genes was calculated with the MEGA program. Stop codons were excluded from the calculation, because they are not linked to a tRNA family. Analogously, the start codons were omitted, because different codons determine the same formyl-Met as starting amino acid^{47 (link), 48 (link)}. The abundance of each codon family was expressed as number of codons per thousand codons (CDSpT). The skews computations as well as other statistical calculations were performed with the spreadsheet Microsoft Excel (Microsoft™).

Amplified PCR products of MG-targeted gene-positive extracts were submitted to determine the sequence. Partial mgc2 gene sequences (Accession Numbers KX268616–KX268632) from 16 Thai MG isolates had been submitted to GenBank in a previous study²⁰ (Table 3.) All sequences were analysed with the Editseq program (Lasergene, DNASTAR Inc., USA), and a consensus was constructed with the Seqman program (Lasergene, DNASTAR Inc., USA). Thai MG isolates and reference gene sequence data were aligned to construct a phylogenetic tree in Bionumeric version 7.6 software (Applied Maths, Sint-Martens-Latem, Belgium). Cluster analysis was performed with the UPGMA method. The similarity coefficients of Thai MG isolates and reference strains were determined from multiple sequence alignments.