Clc main workbench v7

We used CLC Main WorkBench v7 (Qiagen, Aarhus, Denmark) to calculate GC frequencies of all nucleotide sequences encoding centipede venom proteins and peptides published by Jenner et al.^{23 (link)}. Descriptive statistics were calculated with GraphPad Prism v8.4.1 (GraphPad Software, La Jolla California USA, www.graphpad.com), and are available as Supplementary Data 3.

The sequences were examined using CLC Main Work Bench v.7 (www.qiagenbioinformatics.com) to detect base calling conflicts. The forward and reverse sequences were aligned to generate consensus sequences, then compared to available sequences in the NCBI database by using BLAST tools. A cutoff point of 95% of sequence similarity [26 (link)–28 ] was used to identify species for each bushmeat sample, however, the top hit (in most cases > 98% similarity) was recorded as the species of origin.
All statistical analyses were performed in R v3.4.4 [29 ]. The kappa2 function of the IRR package was used to determine concordance between seller-reported and laboratory-confirmed results[30 ]. A Chi-squared test was performed to test whether the match/mismatch percentages were similar between different reported species using the prop.test function in R. The prop.test function was also used to perform pairwise analysis of the individual species (seller-reported versus laboratory-confirmed) [16 (link)]. One-sample t-test power analysis was performed to calculate the sample size with the following assumptions: power = 0.95, Significance = 0.05, an Effect size of 0.25.

Representative genomes from individual Burkholderia, Paraburkholderia, Caballeronia and Robbsia species were downloaded from the NCBI RefSeq database (accessed Feb. 2017). In addition, genomes from strains LMG 28154^T, TSV85 and TSV86 were added to the database. Nucleotide and amino acid sequences of annotated CDS were extracted and searched against a database of 40 nearly universal, single copy gene markers using the FetchMG program with the –v flag to retain only the best hits (Sunagawa et al., 2013 (link)). Twenty-three COGs present in all genomes were extracted, aligned with Clustal Omega and the resulting alignments were concatenated using the AlignIO utility of Biopython (Cock et al., 2009 (link)). The concatenated alignment was trimmed with TrimAl (Capella-Gutierrez et al., 2009 (link)) to remove columns with >10% gaps and poorly aligned sections of the alignment were removed manually in CLC Main Workbench v.7.7 (Qiagen, Aarhus, Denmark). A concatenated nucleotide alignment of 15092 bp was used to build a phylogenetic tree with FastTree, using the GTR CAT model (Price et al., 2010 (link)). The tree was edited in iTOL (Letunic and Bork, 2016 (link)).

CnFGV1 genomic dsRNA was extracted using the Double-RNA Viral dsRNA Extraction Mini Kit (iNtRON Biotechnologies, Seongnam-Si, Korea) and electrophoresed on a 1.5% (w/v) agarose gel. Potentially present fungal DNA and ribosomal RNA were eliminated treating the extracts with both enzymes dsDNase and S1 Nuclease (Thermo Fisher Scientific, Waltham, MA, USA). A subset of three dsRNA extracts (M10535, M10544, and M10545) was subjected to RNA-seq using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina MiSeq v2 (Microsynth AG, Balgach, Switzerland). De novo assembly of reads was carried out using Trinity v2.6.5 [39 (link)]. The obtained contigs were aligned using CLC Main Workbench v7 (CLC bio, Qiagen Digital Insights, Hilden, Germany) and subjected to searches using the ORFfinder resource of NCBI (https://www.ncbi.nlm.nih.gov; accessed on 22 July 2021) and the BLASTp suite of the UniProt portal (v 2.9.0+; https://www.uniprot.org; accessed on 22 July 2021).

Sequencing was carried out in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, CA, United States) and the resultant chromatograms analysed and aligned using CLC Main Workbench v7.7 (CLCBio, Aarhus, Denmark) and by MEGA software v.6.06 [20 (link)]. MEGABLAST, BLASTn and BLASTp algorithms were used for nucleotide and putative protein similarity searches through GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) [21 (link)]. Nucleotide and amino acid sequence alignments were generated in CLUSTAL W, implemented in the BioEdit software [22 (link)]. Phylogenetic and molecular evolutionary analyses were performed with the maximum likelihood method using Tamura-Nei and Jones-Taylor-Thornton algorithms for nucleotide and amino acid sequences, respectively. These were determined as optimal algorithms using Find best DNA/protein- substitution model tools implemented in MEGA v.6.06.