The progress of next-generation sequencing technology has caused a technological breakthrough at the whole-genome level in a large number of species1. Especially, RNA-sequencing (RNA-Seq) has enabled us to take a snapshot of global gene expression in various organs and cells, regardless of any information of a reference genome. RNA-Seq outputs are digital data that can be uploaded to the public database, and sequence information can be shared worldwide.

RNA-Seq analysis also allows us to compare the biological similarity of embryonic stem cells (ES cell) with induced pluripotent stem (iPS) cells. In general, stem cells can be classified into two major subtypes: nave and primed states2,3. ES/iPS cells at nave state of pluripotency, reflect the characteristics of pre-implantation embryos and are applicable in rodents, which contribute to chimeras and germ line4,5. The growth of nave stem cell depends on the activation of LIF (Leukemia Inhibitory Factor) signaling, whereby the cell forms colonies with three-dimensional shape.

On the other hand, primed cells have the characteristics of post-implantation embryos and rarely contribute to chimeras and germ line. In brief, primed cells are already at a more differentiated stage compared to nave cells. Primate ES/iPS cells were conventionally believed to be established in primed state6,7. However, recent publications have demonstrated a reliable method for transforming human ES cells from primed to nave state8,9. Transcriptome analysis using RNA-Seq played an important role in identifying the cellular characteristics reported in those articles.

In the case of pig iPS cells, the status of the cellsnave or primedremains inconclusive since the pluripotent genes have a wide variety of phenotypes. To understand the biological variety of pig iPS cells, multiple datasets of global gene expression profiling would be needed. Although a significant number of reports on the establishment of pig iPS cells have been published10,11,12,13,14,15,16,17,18,19,20, expression profiling data in Sequence Read Archive (SRA) database are quite limited. Therefore, detailed biological features of pig iPS cells need to be addressed with whole expression profiling.

In our previous publication, we had reported that pig iPS cell, derived from six reprogramming factors, has more advantageous than that derived from four factors. Especially, the expression of six reprogramming factors was suitable for X chromosome re-activation21, which is one of the mile-stone characteristics of nave-type stem cells. Our previous data using Ion Torrent sequencing also proved that the expression of six reprogramming factors was more advantageous to activate various pluripotent genes. Although the data obtained from Ion Torrent is suggestive, at least 20M reads would be necessary to obtain a quantitative evaluation of the relatively low-expressing genes. The data obtained in our previous publication seem insufficient in terms of the number of sequencing reads required to conclude. This situation led us to detect the global expression profile of pig iPS cells, derived from the expression of six reprogramming factors, using Illumina short-read sequencer, HiSeq 2500. Currently, there are no publicly available dataset of six factor-derived pig iPS cells using Illumina sequencing platform.

The aim of this study was to clarify the difference of mRNA expression profiles between pig iPS cells derived from six and four reprogramming factors. We found relevant submitted data from two research groups on pig iPS cells with four reprogramming factors, in SRA22,23. We could compare ours with these gene expressions since both datasets were obtained with Illumina sequencer. In this study, we describe the detailed expression profile of pig iPS cells derived from four and six reprogramming factors. Multiple analyses demonstrated that the pig iPS cells derived from six factors formed independent clusters compared to those derived from four factors, and were distant from fibroblasts. Furthermore, we detected that the expression levels of various nave-specific genes were relatively elevated in pig iPS cells derived from six factors. Our data set would contribute to the understanding of biological differences between the iPS cells derived from six and four reprogramming factors, and provide the scientific explanation of how diversity of pluripotency-related genes related to the process of animal evolution.

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Global transcriptome analysis of pig induced pluripotent ...