Sanger Sequencing of Lyssaviruses
Sanger dideoxy terminator sequencing was developed by Fred Sanger and colleagues in 1977 and became the most widely used method for virus characterization in the last three decades. More recently, Sanger sequencing has been supplanted by Next-Generation Sequencing (NGS) methods, for the determination of viral genomes. However, the Sanger method continues to be employed for the sequencing of lyssavirus polymerase chain reaction (PCR) products to facilitate virus typing, molecular epidemiological, or evolutionary studies. Sequencing overlapping PCR products (or “Walking the Genome”) by the Sanger method may still be cost effective for small-scale virus genome studies. This chapter outlines the protocols employed to prepare and sequence lyssavirus PCR products by the Sanger method.