Illumina HiSeq 2000 sequencing; [KIL-d] experiments
[KIL-d] experiments
WT (non-[KIL-d]) M0 or [KIL-d] M0 haploid cells were crossed with WT K+R+ haploid cells. The resulting diploid was cultured for two days and plated on YPAD plate at pH4.7. The diploid cells are defined as âG1â yeast, which exclusively harbor K+R+ virus and exhibit K+ phenotypes (Figures 4A and 4B). The G1 yeast was successively passaged on YPAD plates at pH4.7 and killer phenotypes of the resulting yeast cells in a distinct number of passage (generation) were examined by the killer assay. The yeast cells were passaged until K- phenotypes were clearly observed. Viral RNA and mRNA were extracted from distinct generations of yeast and following RT-PCR was performed as described above. The cDNA was preamplified by PCR with PrimeSTAR Max (Takara) using the primers which contain a tag sequence (Illumina) for identification of each sample in the multiplexed samples and an adaptor sequence for deep sequence analysis. Primers used in this study were listed in Table S6. Deep sequence analysis of the libraries was performed on Illumina HiSeq2000 in Hokkaido System Science CO., Ltd or Illumina HiSeq2500 in RIKEN BSI Research Resources Center (RRC). The total number of sequence reads in each sample was approximately 5,000,000. All data were processed and analyzed using CLC Genomics Workbench (CLC bio, Katrinebjerg, Denmark). Sequence data were referred to the K1, L-A, 20S RNA viral genome sequences or the S. cerevisiae TUB1 sequence. In the analysis of sequence data, each nucleotide data whose quality value was less than 40 (error probability =0.0001) was eliminated in order to increase the reliability of the sequence data. For calculation of the rate of accumulation of new mutations in the M1 viral genome of WT and [KIL-d] yeast, the sequence data at the nucleotide position 229 in the killer toxin gene were removed. The rate of de novo mutations in M1 virus, 20S virus and yeast TUB1 genes in Figures 4 and S11 was determined by the deep sequence data from independent colonies of distinct generations (G1-G12) of yeast.  Viral RNA and mRNA in yeast were isolated by phenol extraction of whole yeast cells as described previously (Schmitt et al., 1990). Briefly, AE buffer (50mM Sodium acetate, 10mM EDTA) and glass beads were added to yeast cell pellets and yeast cells were broken by multi vortex. RNA was extracted by phenol extraction using phenol saturated with citrate buffer, pH4.3 and chloroform. The extracted RNA was concentrated by propanol precipitation. The cDNA was preamplified by PCR with PrimeSTAR Max (Takara) using the primers which contain a tag sequence (Illumina) for identification of each sample in the multiplexed samples and an adaptor sequence for deep sequence analysis.