Tacaribe Virus Defective Viral Genome Sequencing Reads

Defective viral genomes (DVGs) are byproducts of RNA replication that are produced during infection with diverse RNA viruses. In order to gain insight into DVG generation during arenavirus infection, we serially passaged Tacaribe virus (TCRV) at a high multiplicity of infection, which led to the accumulation of smaller sub-genomic products at the expense of full-length genomes. These DVGs from 3 independent experiments were amplified using a set of RT-PCRs using the SuperScript III Reverse Transcriptase (Invitrogen) with a universal L and S segment genome end primer (CGCACAGTGGATCCTAGGC) and iProof High-Fidelity PCR Kit (Bio-Rad) with segment-specific genome end primers (S segment Fwd: GGCAAATTGTCTAACTCTTTCACTGAG, S segment Rev: CCTAGGCATTTCTTGAC-CATATTTGC, L segment Fwd: ATCCTAGGCGGCACTTGACC, L segment Rev: CCTAGGCGTTAC-GTGCACTC). To amplify deletion DVGs, both forward and reverse primers were used for amplification of the respective segment, while for detection of copyback DVGs PCR was run with either only the forward or only the reverse primer. PCR products were purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel), pooled, and prepared for nanopore sequencing using the 1D2 kit according to the manufacturer's protocol (Oxford Nanopore Technologies, version LSD_9032_v11_revO_23Mar2017). A 16 hour sequencing was performed on a MinION sequencer containing a FLO-MIN107 R9 flow cell using the MinKNOW software (version 2.2.15) with integrated live base-calling.