A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library

Table 1 Recombination and mutation rates

RNA was extracted from either an equal mix of homozygous WT and MK viruses or from an equivalent amount of heterozygous virus and reverse transcribed using increasing amounts of RNA as template using SSIII reverse transcriptase (RT), engineered to have minimal RNase H activity. In a parallel experiment RNA was also reverse transcribed using AMV with intact RNase H activity. Following first strand synthesis, cDNA was diluted as appropriate and amplified using 27 or 35 PCR cycles. Replicates containing SYBR Green 1 dye were used to ensure the reaction was stopped while in the log linear phase (27 cycles) or at plateau (35 cycles). Replicate samples were pooled and prepared for 454 NGS sequencing. Recombination and mutation rates are expressed as rate per 1000 nucleotides. The 95% confidence interval lower and upper boundaries, as determined by bootstrapping, are shown for each estimate.

ISSN: 1743-422X