From ABI Sequence Data to LASERGENEs EDITSEQ

This chapter describes the analysis and assembly of sequence data generated by the Applied Biosystems (ABI, Foster City, CA) automated sequencer after sequencing PCR products using Taq dye terminators (e.g., with the PRISM DyeDeoxy Terminator cycle sequencing kit). We use Taq dye terminator chemistry rather than dye primer sequencing because we are currently interested in sequencing PCR products of viral genomes amplified from clinical specimens (1 ,2 ). We prefer to sequence PCR products directly rather than after cloning them to avoid seeing unrepresentative sequences. To be able to look at the distribution of sequences in a population (the quasispecies), we dilute the starting specimen until amplification is achieved from a single molecule (3 ). Alternatively, if the sequence population is relatively homogeneous, sequencing from a bulk PCR amplification will be accurate since Taq misincorporation errors will be effectively diluted out.