Novaseq 6000 s4 lane

DNA was extracted from FTA cards in a designated pre-PCR BSL-2 laboratory using the Qiagen PowerSoil kit (Qiagen; cat. no. 47014). Every batch of 8–24 sample extractions included a negative control. Samples with discoloration after the wash buffer step in the protocol were eluted and re-bound to filter columns with binding buffer for one to three additional washes.
Genomic libraries were constructed using the Illumina DNA Prep kit (Illumina, cat. no. 20018705). Duplicate samples were randomly selected or combined for separate library builds for the Belize colony, the aggregated zoo populations and the experimentally merged colony (electronic supplementary material, table S1). Extraction negatives had negligible quantities of DNA as quantified by Qubit readings (ThermoFisher Scientific, cat. no. Q32851), and were combined to make per-population negative libraries. A separate library negative was also constructed. Overall, 145 samples, including samples, duplicates and listed negative controls, were sequenced on a NovaSeq 6000 S4 lane (Illumina), resulting in approximately 1.75 billion 150 bp paired-end reads. Because we used DNA extraction without reverse transcription, our microbiome analyses do not include RNA viruses.

Sequencing libraries for Veg2 and Veg6 were generated using EquiPhi polymerase (Thermo Fisher Scientific, Waltham, MA, USA) for rolling circle amplification (RCA) and the Nextera XT kit (Illumina, San Diego, CA, USA) with unique dual index sequences for library preparation [50 ]. The protocol was modified to include two RCA reactions for each sample. Each RCA reaction was diluted to 5 ng ml⁻¹, and 2 µl of each reaction were combined. The combined RCA reactions were diluted to 0.2 ng/µl (1 ng total in 5 µl) and used to construct two technical replicate libraries. Libraries of the inoculum plasmids for ACMV and EACMCV (1 ng of plasmid DNA in 5 µl) were also prepared using the Nextera XT kit. The libraries were pooled in equimolar amounts and sequenced on an Illumina NovaSeq 6000 S4 lane to generate 150 bp, paired-end reads.

Muscle samples were homogenized using a Tissuelyzer (Qiagen, Valencia, CA) in the presence of Trizol reagent. The RNA was extracted using Qiagen RNAeasy mini kit (Qiagen N.V., Valenca, CA, United States) according to the manufacturer’s protocol. The RNA sample purity was evaluated by determining A260/230 nm and A260/280 nm ratios using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Santa Clara, CA, USA, 2007). The RNA sample integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) to obtain the RNA integrity number (RIN) values. A total of 35 RNA samples (7 animals × 5 time points) were sequenced at the Génome Québec Innovation Centre (McGill University, Montréal, QC, Canada). Briefly, pooled libraries were prepared and loaded at 225 pM on an Illumina NovaSeq 6000 S4 lane following manufacturer’s protocol. The sequencing run was performed for 2x100 cycles in paired-end mode, generating 100 bp length, paired end sequencing reads. Base calling was performed with RTA v3.4.4 program, and the samples were de-multiplexed to generate sample-wise raw sequence files in fastq format using bcl2fastq2 Conversion Software v2.20.

Total DNA was extracted from the head and thorax for adult samples, and from the whole body for individuals at the nymphal stage as described in Fridi et al., [23 (link)]. DNA sequencing was performed at the GeT-PlaGe core facility (INRAE Toulouse). DNA-seq libraries were prepared according to Illumina’s protocols using the Illumina TruSeq Nano DNA HT Library Prep Kit. Briefly, DNA was fragmented by sonication and adaptors were ligated for sequencing. The libraries were amplified for eight PCR cycles and quantified by qPCR using the Kapa Library Quantification Kit. Library quality was assessed using an Advanced Analytical Fragment Analyzer. DNA-seq experiments were performed on an Illumina NovaSeq 6000 S4 lane using a paired-end read length of 2 × 150 pb with the Illumina NovaSeq 6000 Reagent kits. Sequence reads were made publicly available after deposition on SRA (www.ncbi.nlm.nih.gov/sra/PRJNA1044268).

Libraries were normalized and pooled and then denatured in 0.02 N NaOH and neutralized using HT1 buffer. The pool was loaded at 175 pM on an Illumina NovaSeq 6000 S4 lane using Xp protocol as per the manufacturer’s recommendations. The run was performed for 2 × 100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level. Base calling was performed with RTA v3. Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate FASTQ reads.