Most viral pathogens are single-stranded RNA including SARS-CoV-2, the virus behind the COVID-19 pandemic. This protocol describes how to convert RNA into cDNA libraries for downstream use in NGS Target Enrichment. The protocol begins with creating cDNA from extracted/purified RNA using commercially available reagents. The cDNA is then converted to Illumina TruSeq-compatible libraries using the Twist Library Preparation EF Kit 2.0 with Enzymatic Fragmentation and Unique Dual Indices (UDI). The resulting cDNA libraries are compatible with Twist Target Enrichment Protocols. Synthetic viral RNA controls, such as the Twist synthetic SARS-CoV-2 RNA controls, can be spiked into reference RNA samples to serve as a positive control.