Exploring the Impact of Primer Length on Efficient Gene Detection via High-Throughput Sequencing

Reverse transcription (RT) is a crucial step in most RNA analysis methods, including quantitative PCR and high-throughput RNA sequencing. Optimizing protocols for this initial stage is vital for effective target detection, particularly when working with limited input RNA. Several factors, such as input material quality, priming strategy, and reaction conditions, influence RT efficacy. However, the impact of RT primer length on gene detection efficiency remains largely unknown. In this study, we conducted a systematic analysis to explore the influence of primer length on gene detection using RNA sequencing. We generated RNA-seq libraries from SARS-CoV-2 Delta variant infected Vero cell line RNA samples, employing random RT primers of varying lengths: 6, 12, 18, or 24 nucleotides.