Shotgun sequencing of the negative-sense RNA genome of the rhabdovirus Maize mosaic virus

The maize-infecting nucleorhabdovirus, Maize mosaic virus (MMV), was sequenced to near completion using the random shotgun approach. Sequences of 102 clones from a cDNA library constructed from randomly-primed viral RNA were compiled into a 12,133 nucleotide (nt) contig containing six open reading frames. The contig consisted of 97 sequences averaging 660 bp in length. The average sequence coverage was six-fold, and 93% of the contig had sequence reads covering both strands. The remaining sequence was derived from single (5%) or multiple (2%) reads on the same strand. Three of the six ORFs showed significant similarities to the deduced protein sequences of the nucleocapsid, glycoprotein and polymerase sequences of other rhabdoviruses. The predicted gene order of the MMV genome was 3′-N-P-3-M-G-L-5′. Shotgun sequencing of the MMV genome took approximately 127 h and cost $ 0.38 per nt (including labor), whereas the primer walking approach for sequencing the 13,782-nt MFSV genome [Tsai, C.-W., Redinbaugh, M.G., Willie, K.J., Reed, S., Goodin, M., Hogenhout, S. A., 2005. Complete genome gequence and in planta subcellular localization of maize fine streak virus proteins. J. Virol. 79, 5304-5314] took about 217 h and cost $ 0.50 per nt. Thus, the shotgun approach gave good depth of coverage for the viral genome sequence while being significantly faster and less expensive than the primer walking method. This technique will facilitate the sequencing of multiple rhabdovirus genomes.