Amplifying and Sequencing CHIKV RNA from Mosquitoes

Amplicon libraries were prepared as previously described [45 (link)]. Briefly, CHIKV RNA isolated from bodies and saliva was amplified by nine separate RT-PCR reactions using a high-fidelity RT-PCR kit and the primers described in Riemersma et al., 2019 [45 (link)]. The nine overlapping cDNA amplicons spanning from the 5’ to 3’ untranslated regions (UTR), excluding the first 14 nucleotides of the 5’ UTR and the last 55 nucleotides of the 3’ UTR, were pooled at equimolar ratios and enzymatically fragmented to approximately 150 bp. Illumina sequencing libraries were prepared with the fragmented cDNA using a NEBNext Ultra II DNA library prep kit and NEBNext Multiplex Oligos (New England Biolabs). Libraries of P4 infectious plasmid DNA with (pY_PCR) and without (pY) RT-PCR amplification, and P4 in vitro transcribed RNA were used as sequencing controls. An unrelated library (WNV cDNA) was also included as a control for index hopping with Illumina HiSeq 4000 sequencing [56 ]. All libraries were sequenced in parallel with paired-end 150 reads on a single flow cell lane of an Illumina HiSeq 4000 instrument at the UC Davis DNA Technologies Core. Raw fastq files are available from NCBI Sequence Read Archive under BioProject entry PRJNA541092.