Somatic mutations during reprogramming to pluripotency revealed by massively parallel sequencing of iPS cell whole genomes

Abstract

Background: Despite induced pluripotent stem cell (iPSC) reprogramming success, the whole genome sequence of iPSC has rarely been explained at high throughput. As iPSC can potentially replace embryonic stem cells for clinical applications as a therapeutic agent it is important to understand genomic changes occurring during the reprogramming process. As described by Nagy A. et al. iPS cell fate is uncertain during a timeframe from Day 5 to Day 15, and reprogramming to pluripotency is unavoidable after Day 16. In order to assess mechanisms necessary to the reprogramming and arising genomic aberrations, we studied variants arising before, within and after the timeframe of uncertain cell fate in iPSC generated from mouse fibroblasts. Method: We generated a whole genome sequence with the next generation sequencer Illumina HighSeq 2000 at a high coverage of 35.77X in average of 5 samples

primary iPSC, three intermediate stages of reprogramming at Day 0, Day 11 and Day 18, and secondary iPSC. We then performed single nucleotide polymorphism and short insertion-deletion genotyping with our GMI caller and compared our method with two different algorithms, Samtools and MAQ. Subsequent gene ontology was performed with Ontologizer 2.0. Results: We identified more than 5 million single nucleotide polymorphisms (SNPs) per sample and our results showed that the number of non-synonymous SNPs, unique SNPs per sample compared to 1iPSC and the number of indels reached a maximum at D11 and then decreased until 2iPSC. Thus as reported by Ji J. et al. we found that cells reprogramming to pluripotency have an increased number of somatic coding mutation from D11 to 2iPSC. Gene Ontology of somatic non-synonymous SNPs occurring at the end of the reprogramming revealed that biological process such as gene expression (p=9.05 x 10-3), cell differentiation (p=9.38 x 10-3) and cell proliferation (p=7.79 x 10-3) are affected by coding mutations. These results suggest that genes involved in these biological processes might have a role in the reprogramming to pluripotency. Conclusion: The timeframe of uncertain cell fate is a critical time limit for the reprogramming process. Like previously reported by Ji J. et al. our results showed an elevated coding mutation rate during the reprogramming. Finally, gene ontology showed that coding mutations are affecting important biological process at the end of the reprogramming such as cell differentiation, the immune system and gene expression. Therefore, we encourage further characterization of these variants to understand mechanisms involved during the timeframe of uncertain cell fate.