the parental cell line hek293 showed a notable difference in transcriptome profile compared to all the other cell lines along the first principal component ( pc1 ). these results indicate a genomic divergence of the clonal lineages compared to the parental hek293 and suggest the presence of similar transcriptomic traits between hek293 progeny cell lines individually selected for during the isolation of each clone. hierarchical clustering of the cell lines based on snvs gave a slightly different trend compared to the transcriptomic comparison. a different pattern of overall clustering was observed, with the original hek293 and 293e cell lines separated from the rest on a separate branch and the three suspension cell lines grouped on a second branch together with 293t. however, 293 - f and freestyle 293 - f were, as expected, the most similar cell lines also in this comparison ( supplementary fig. s1 ). the overall number of genomic variations was similar between the cell lines and variations located to similar genomic regions ( supplementary fig. s1 ). moreover, the ratio between missense and synonomous snvs in all cell lines ranged between 0. 866 and 0. 879, where the original hek293 strain had the lowest ratio ( supplementary fig. s1 ). pairwise comparisons of snvs and indels between hek293 and each progeny cell line showed that the highest number of high ( variants expected to have a disrup tive impact on the protein, for example protein truncation or loss of start / stop codon ) and moderate ( non - disrup tive variant that might change protein effectiveness, for example a missense variant ) snvs and indels were seen for the two adherent cell lines 293e and 293t ( supplementary fig. s1 ). genes with high impact snvs in progeny cell lines compared to the parental hek293 can be found in supplementary table s2. amongst these, five genes had acquired high impact snvs ( ppp2r4, c9orf43, ctb - 47b11. 3, cyfip2 and sgcd ) in all progeny cell lines compared to the parental strain. figure 1comparisons of genomic and transcriptomic profiles of hek293 cells showed taxonomic divergence between parental hek293 and progeny cell lines. ( a schematic overview of the lineage relationship of the six hek293 cell lines used in this study. blue dots represent adherent cells whereas grey dots represent suspension cell lines. ( genomic comparison between hek293 cell lines based on spearman correlation coefficients of read counts. darker blue color indicates higher correlation. ( sample - to - sample comparison between transcriptomes illustrated by a heatmap and hierarchical clustering of taxonomical divergence between samples. darker blue color indicates shorter euclidean distance between samples and more similarity. ( pca plot showing the separation in expression pattern between samples. ( rna expression levels ( in deseq2 median of ratios ) and standard deviations ( n = 3 ) of stably integrated viral genes ( ebna - 1, large t, e1a and e1b ) in hek293 cell lineages determined by rna sequencing. comparisons of genomic and transcriptomic profiles of hek293 cells showed taxonomic divergence between parental hek293 and progeny cell lines. ( a schematic overview of the lineage relationship of the six hek293 cell lines used in this study. blue dots represent adherent cells whereas grey dots represent suspension cell lines. ( genomic comparison between hek293 cell lines based on spearman correlation coefficients of read counts. darker blue color indicates higher correlation. ( sample - to - sample comparison between transcriptomes illustrated by a heatmap and hierarchical clustering of taxonomical divergence between samples. darker blue color indicates shorter euclidean distance between samples and more similarity. ( pca plot showing the separation in expression pattern between samples. ( rna expression levels ( in deseq2 median of ratios ) and standard deviations ( n = 3 ) of stably integrated viral genes ( ebna - 1, large t, e1a and e1b ) in hek293 cell lineages determined by rna sequencing. the hek293 cell line was originally immortalized by the random integration of viral genomic dna of adenovirus 5, which includes the e1a and e1b genes. in this study, overall high mrna levels of e1a and e1b were observed in all hek293 cell lines ( supplementary fig. s2 ). a comparison of mrna levels of the viral element e1a showed significantly ( p < 0. 05 ) higher expression in hek293 compared to both 293t and freestyle 293 - f. in addition, both 293e and 293 - h had significantly higher expression than 293t, 293 - f and freestyle 293 - f. further, 293 - f had significantly higher expression than freestyle 293 - f. ( supplementary fig. s1 ) the analysis of the viral element e1b showed that 293 - f had significantly higher expression ( p < 0. 05 ) than 293 - h and freestyle 293 - f ( supplementary fig. s1 ). as expected, the gene expression of large t and ebna - 1 was detected in 293t and 293e, respectively ( fig. 1e ). interestingly, expression of the large t antigen was also observed in 293e, which is not reported by the supplier ( atcc ). the presence of a truncated version of large t in the 293e genome was confirmed by de novo assembly of all reads not mapping to the human reference genome ( supplementary fig. s2 ). tracing the origin of the 293e cell line, the large t expression of 293e may be derived from the prsvneo plasmid that was used to co - transfect hek293 cells along with the pcmv - ebna plasmid for the generation of the stable ebna - 1 expressing clone ( 293c18 ) by geneticin ( g418 ) selection. the prsvneo plasmid contains a truncated version of the large t gene ( according to the addgene vector database ), which aligns perfectly with the truncated large t sequence found in the 293e genome ( supplementary fig. s2 ). in order to evaluate the genomic variation between hek293 and its derivatives further, overall genomic copy number variation of all progeny cell lines compared to the parental hek293 was performed. a comparison of gained and lost regions on all chromosomes between all cell lines can be found in supplementary fig. s3 and table s3. interestingly, a conserved pattern of copy number gain or loss of large regions has occurred on several chromosomes of all hek293 progeny cells compared to hek293, whereas other changes are more local or cell line specific. for instance, on chromosome 13, a region of > 15 mb has been amplified in all cell lines compared to the parental hek293 strain ( fig. 2a ). all elements with copy number gain of > 1 log2 fold - change common to all progenitor cells are located in this region ( supplementary table s3 ). amongst these, four out of seven protein - coding genes ( bora, mzt1, pibf1 and klhl1 ) belong to the cytoskeleton gene set ( go: 0005856 ). on chromosome 18, there is a conserved pattern of copy number loss of most of the chromosome sequence for all progeny cell lines compared to the parental hek293 strain, with the exception of a high degree of copy number gain ( > 0. 8 log2 fold - change ) of a region close to the centromere for all cell lines except 293e ( fig. 2a ). within the region of conserved gain are several genes encoding cell adhesion molecules within the desmocollin ( dsc ) and desmoglein ( dsg ) subfamilies, belonging to the cell – cell adhesion gene set ( go: 0098609 ). when analyzing more local copy number variations between progeny cell lines and the parental strain, some interesting loss or gain of full or partial elements compared to the parental hek293 were identified. for instance, copy number loss was observed for the fumarate hydratase ( fh ) gene, which has previously been reported to have lost several gene copies in hek293 and hence been hypothesized to play a role in the phenotypic transformation of hek293.