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Figure 5 | Virology Journal

Figure 5

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

Figure 5

Occurrence of transfection induced recombination. Homozygous and heterozygous viruses were produced by transfection of HEK293T cells as described in the methods. RNA was extracted from an equal mix of WT and MK virus or from an equivalent amount of heterozygous virus. In parallel reactions SSIII was used to reverse transcribe 160 ng as input template. Resulting cDNA was diluted and amplified using an optimised 2-step PCR and 27 cycles to minimise PCR-induced recombination [1]. Any recombination occurring in the mix of homozygous viruses can only be a consequence of RT-PCR, PCR and/or 454 sequencing (laboratory induced recombination). Recombination in the heterozygous virus reflects both laboratory induced recombination and transfection induced recombination. Recombination and mutation rates are expressed as rate per 1000 nucleotides and the confidence intervals as estimated via bootstrapping are shown. There was no significant difference between these estimates.

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