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Protocol publication

[…] Library sizes were determined empirically by microfluidic analysis on an Agilent 2100 Bioanalyzer and quantified using the Quant-iT dsDNA kit with broad range standards (ThermoFisher Scientific, Q-33130). Samples were pooled together at equimolar quantities and sequenced twice using the Illumina MiSeq (2x300) cycle kit with version 3 chemistry. Using the Illumina indices, the data were demultiplexed and the runs combined to assign the data to individual samples. All other procedures were followed according to the manufacturer’s instructions., Sequences were aligned to the Solenopsis invicta reference genome downloaded from using the Burrows-Wheeler Aligner (bwa-0.7.5a), a software package for mapping low-divergent sequences against a large reference genome []. The S. invicta unmapped reads were selected and converted to FASTA format using NextGENe-2.3.4 (SoftGenetics, State College, PA). Each read was then filtered and retained if the median score was ≥ 20 and base number ≥ 25. Unmapped and filtered individual MiSeq sequences were analyzed using BLASTX [] against the curated Swiss Protein database (; download date 11/14/2014). Sequences returning an expectation score less than 10−5 were tabulated., Based on the BLASTX results, each sequence was annotated and assorted taxonomically. The sequences were binned into the following groups: Animal, Plant, Fungi, Bacteria, Archaea, Phage, and Non-phage virus. Also at this stage, sequences exhibiting identity to Enterobacteria phage phiX174, an internal control for Illumina processing [] were removed and not considered in subsequent analyses., Non-phage virus sequences from each library identified from the BLASTX analysis were assembled using the CAP3 algorithm [] in the Vector NTI ContigExpress program (Invitrogen, Carlsbad, CA). Sequences from phage were not assembled because they infect bacteria and would not be expected to infect fire ant cells. Contiguous sequences (contigs) and remaining singletons were matched to the genomes of known fire ant viruses (Solenopsis invicta virus 1 [SINV-1, GCF_000854925.1], SINV-2 (GCF_000870805.1), SINV-3 (GCF_000881215.1), SINV-4 (MF_041808.1), and SiDNV (GCF_000912895.1)). Those sequences matching known fire ant viruses (≥ 95% identity) were binned according to virus species and excluded from further analysis. Virus unmatched sequences/contigs were re-analyzed by BLASTX and sequences returning […]

Pipeline specifications

Software tools BWA, NextGENe, BLASTX, CAP3