454 gs junior platform

For each sample, genomic DNA was extracted from 500 mg of soil using the FastDNA^TM SPIN kit for soil (MP Biomedicals, USA) following the manufacturer’s instructions. For fungi, the nuclear ribosomal internal transcribed spacer (ITS) region was amplified using ITS1F and ITS4 primers [30 (link)], while for bacteria, the 16S ribosomal DNA (16S) region was amplified using 27F [31 (link)] and 518R primers [32 ]. Amplicon libraries were prepared using primers with a 454 pyrosequencing adaptor and multiple identifier (MID) tag. The PCR program was as follows: 5 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 55°C, and 40 s at 72°C; and a final extension step of 5 min at 72°C. PCR products were confirmed using gel electrophoresis and purified using the HighPure^TM PCR Product Purification Kit (Roche, Germany). Individual PCR products were quantified using a NanoDrop spectrophotometer (Thermo, USA) and separately pooled for bacteria and fungi. Pyrosequencing was performed with a 454 GS Junior platform (Roche, USA) at ChunLab (Seoul, Korea). DNA was sequenced only in the reverse direction, and data were deposited in the NCBI Sequence Read Archive (SRP046049).

Deep bisulfite sequencing was performed on the 454 GS junior platform (Roche) as described previously26 (link). Data analysis was done using the Amplikyzer software27 and results are depicted as methylation heatmaps showing single CpG sites in columns and single reads in rows. The percentage of overall methylation is given in Supplementary Table S6. Amplicon-specific tagged primer sequences are given in Supplementary Table S8, as well as an example for a MID-primer pair. For the Epi-Pluri-Score analysis, pyrosequencing of three CpGs of interest (cg 23737055 in ANKRD46, cg22247240 in VRTN, cg13083810 in POU5F1) was performed and analyzed as described by Lenz et al.13 (link). The Epi-Pluri-Score is calculated as the difference of β-value(ANKRD46) minus β-value(VRTN). A positive value is indicative of pluripotent cells, a negative value of differentiated cells. The Epi-Pluri-Score is plotted against the β-value measured for POU5F1. In the plot, reference sets of 265 pluripotent and 1,951 somatic cells are indicated as clouds. Primer sequences are listed in Supplementary Table S8.

The genomes of the ancestor R0 and the evolved cell population R7 were resequenced using a next-generation sequencer (Junior, Roche). Genomic DNA was purified with a Wizard Genomic DNA Purification kit (Promega) and fragmented using a DNA shearing system (Covaris), according to the manufacturer’s instructions. Whole-genome shotgun sequencing by the 454 GS Junior platform (Roche) was performed according to the manufacturer’s instructions. The sequence reads were assembled using Newbler 2.7 and aligned using the GS Reference Mapper software (ver. 2.6; Roche). Approximately 99% of the reads in each dataset (DRA003743, DDBJ) were uniquely mapped to the MDS42 genome (AP012306, DDBJ). The mutations were analyzed using the GS Reference Mapper software. The candidate mutations were further determined by Sanger methods of the genome samples subjected to a resequencing analysis using a genetic analyzer (ABI PRISM 3100, Applied Biosystems). Sequencing was performed to both the purified genomes and the cell pellets of these populations. In addition, the cell populations (R0–R7) acquired in the experiments of growth curves were repeatedly sequenced for further verification.