Enrichment and sequencing of rodent-associated orthohantaviruses

For the HTS libraries, 1 µg of total spleen and liver RNA from one rat from each pet-rat owner was processed using a NEBNext Ultra II RNA library preparation kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions with eight minutes of RNA fragmentation and seven PCR cycles to amplify the resulting cDNA libraries. Amplified cDNA libraries were enriched for hantavirus sequences using a custom-made myBaits tiling array (Arbor Biosiences, Ann Arbor, MI, USA) designed to capture sequences from rodent-associated orthohantaviruses according to the manufacturer’s instructions. Briefly, 1 µg of HTS library was hybridized for 20 hours to biotinylated RNA baits, captured with streptavidin-coated paramagnetic beads, washed extensively to remove non-target sequences, and eluted from the beads through heat denaturation. Enriched sequences were amplified with 10 cycles of PCR using Q5 high-fidelity DNA polymerase (New England Biolabs) and primers annealed to the P5 and P7 library sequences. Enriched and amplified libraries were run on an Illumina MiSeq sequencer using v.3 chemistry for 2 × 300 bp paired-end reads.
Raw sequencing data were processed using Trimmomatic v.0.39 [33 (link)] and mapped against the genome of SEOV strain 201701093/SEOV/Illinois_US/Rat (GenBank accession: MK360784 (S segment), MK360797 (M segment), and MK360803 (L segment)) using the Burrows-Wheeler aligner v0.7.17 [34 (link)]. Mapping statistics were generated using Samtools v.1.10 [35 (link)], and alignments were visualized using IGV v2.9.4 for Linux [36 (link)]. For detection of single-nucleotide polymorphisms (SNPs), Freebayes (a Bayesian genetic variant detector) was used [37 ]. All SNPs with a minimum mapping quality of 5, a minimum count of 3, and a minimum fraction of 0.1 were considered. Consensus sequences for each sample were obtained using BCFtools [35 (link)].