Sequencher program

The identified POU1F1 variant was confirmed and validated with Sanger sequencing and Taqman genotyping. Primer3 (http://bioinfo.ut.ee/primer3/) (Rozen and Skaletsky. 2000 (link)) was used to design the following Sanger sequencing primers; 5′-CCAGGAAAAGTGTGATCGGG-3′ (forward) and 5′-TCCATCTCCTCTGTACGTTTTG-3′ (reverse). Biotools DNA Polymerase (Biotools B&M Labs, S.A.) was used to perform PCR amplification, Sanger sequencing reactions were performed at the Institute for Molecular Medicine Finland (FIMM), and the obtained Sanger sequence data were analysed with the Sequencher program (Gene Codes Corporation). After initial Sanger confirmation, a custom Taqman SNP genotyping assay (ThermoFisher Scientific) was ordered to genotype the POU1F1 variant in larger sample cohorts. The primer and probe sequences for the assay were the following: 5′-TTTGCATTGTTTTAGAAAGAAAATTTGAAACTCAAA-3′ and 5′-CCTTTTTCTTTCATTTGCTCCCACTT-3′ (forward and reverse, respectively), 5′-VIC-ATTCCCCATTACAGCTTT-3′ and 5′-FAM-CTCACCGTAGTCCCCCAT-3′ (reference and variant allele, respectively). The Taqman genotyping reactions were carried out using Biorad’s CFX96 Touch Real-Time PCR Detection System. The utilised canine POU1F1 reference sequences were NM_001006949.1 and NP_001006950.1 for mRNA and protein, respectively.

Primers localized in exon 1 of GULP1 and exon 9, 10, 11, 12, 13 of EPC2 were used to amplify the fusion from cDNA and the products were cloned using the TA Cloning^® kit with pCR™2.1 Vector and One Shot^® TOP10F’ chemically competent E. coli (Thermo Scientific, Dreieich, Germany). Clones were analyzed by Sanger sequencing. A total of 50 ng of the DNA was used for a conventional PCR reaction using 0.1 U of Taq polymerase (Axon Labortechnik, Kaiserslautern, Germany). A total of 0.4 mM of each primer, 200 mM dNTP mix, 1.5 mM MgCl2 as well as 0.2 M betaine and the following PCR conditions: an initial denaturation step at 94 °C for 5 min, 36 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s, and a final extension step at 72 °C for 10 min. PCR products were purified by an enzymatic method using 10 U of exonuclease I (biolabs) and 2 U of shrimp alkaline phosphatase (SAP) for 30 min at 37 °C and 15 min at 80 °C and sequenced by StarSEQ GmbH (Mainz, Germany). The sequences were compared to the reference sequence using Sequencher program (Gene Codes, Ann Arbor, USA).

The obtained PCR products of Anaplasma and Ehrlichia were gel-purified and cloned into a pCR2.1 vector using a TA Cloning kit (Thermo Fisher Scientific, Waltham, MA, USA). Escherichia coli DH5α (TOYOBO, Osaka, Japan) was transformed with the recombinant plasmids. Ten clones were selected randomly for each PCR product and the insert DNA of each clone was sequenced. Other PCR products were purified and sequenced directly. All obtained sequences were assembled and translated into protein sequences using the Sequencher program (Gene Codes Corp., Ann Arbor, MI, USA). Homology searches and species identification were performed using blastn or blastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis of the flaB, glpQ, gltA, p28/omp-1, and p44/msp2 sequences were performed using MEGA 7 with 1000 bootstrap replicates [22 (link)].