Fig. 1. Experimental determination of the unbiased mutation rate of the Thermococcus eurythermalis A501 is challenging because this archaeon has unusual physiology (i.e., obligate anaerobic and obligate hyperthermophilic).

A The preparation of anaerobic high temperature tolerant gelrite plate. After sterilization and polysulfide addition via syringe, the plates are made in an anaerobic chamber. B The incubation of the strain T. eurythermalis A501 at 85 °C in liquid medium. C The initiation of mutation accumulation (MA) by spreading cells from a single founding colony to 100 lines. Plates are placed in an anaerobic jar for incubation in strictly anaerobic condition at 85 °C. D The MA process followed by whole-genome sequencing and data analysis. Single colony of each line is transferred to a new plate for N times (here N = 20). E Base-substitution mutations and insertion/deletion mutations across the whole genome of T. eurythermalis. The dashed vertical line separates the chromosome and plasmid. The height of each bar represents the number of base-substitution mutations across all MA lines within 10 kbp window. Green and red triangles denote insertion and deletion, respectively. The locus tags of the 14 genes with statistical enrichment of mutations are shown.