Fig. 5

Whole-genome sequencing coverage data for examples of wild-type and ΔKU70 K. phaffii cells containing nanochromosomes (Also see Additional file 1: Fig. S7) The y-axes show “normalised coverage” values obtained by dividing the coverage of each base pair by the mean coverage of all base pairs in the respective sample. This allows comparisons, and identification of duplicated segments or deletions. The x-axes show the position (genomic coordinate) of each base pair in its respective chromosome (values in kilobases for native chromosomes). Each nanochromosome is shown as a schematic (colour-coding as in Fig. 1b), drawn to the same scale as the x-axis in each case. * Indicates a centromere. (.) Indicates anomalies attributable to native genome rearrangements (e.g. duplicated loci) or artifacts arising from highly repetitive regions such as found in telomeres. (X) Indicates a ~ 1-kb duplicated region of Chr 2 corresponding to an ORF encoding an unknown protein (inexplicably observed exclusively in ΔKU70 strains transformed with insertion arrays). Red boxes, drawn on the nanochromosome schematics, indicate CEN3 core regions characterised by the signature presence of an extra G:C base pair. Upper panel: For yDA175, on wild-type background, a plot of normalized coverage indicates two-to-four copies of nanochromosomal sequence per cell. This elevated normalised coverage values for nChr 2 suggest multiple copies per cell of its DNA content. The high copy-number for the Chr 3 centromere correlates with a high copy-number of nChr 2 and supports the existence of chimeric multi-centric nanochromosomes (a tri-centric model is suggested in Additional file 1: Fig. S8). This observation is compatible with yDA175 and yDA177 de novo assembly results obtained from long-read WGS (Additional file 3). Middle and lower panels: Both these strains on a ΔKU70 background are consistent with a single copy per cell of its nanochromosome, specifically: yDA253 with a single copy of nChr 2A; and yDA275 with a single copy of nChr 2A.2 (i.e. after the inch-worming proof-of-principle experiment). [(BioProject PRJNA971544)]