Abstract
BackgroundAdvances in sequencing technologies have opened a new era of high throughput investigations. Although RNA-seq has been demonstrated in many organisms, no study has provided a comprehensive investigation of the bovine transcriptome using RNA-seq.ResultsIn this study, we provide a deep survey of the bovine embryonic transcriptomes, the first application of RNA-seq in cattle. Embryos cultured in vitro were used as models to study early embryonic development in cattle. RNA amplified from limited amounts of starting total RNA were sequenced and mapped to the reference genome to obtain digital gene expression at single base resolution. In particular, gene expression estimates from more than 1.6 million unannotated bases in 1785 novel transcribed units were obtained. We compared the transcriptomes of embryos showing distinct developmental statuses and found genes that showed differential overall expression as well as alternative splicing.ConclusionOur study demonstrates the power of RNA-seq and provides further understanding of bovine preimplantation embryonic development at a fine scale.
Highlights
Advances in sequencing technologies have opened a new era of high throughput investigations
The transcriptomes of in vitro fertilization (IVF) embryos showing distinct developmental statuses were previously profiled using microarrays, and few gene expression differences were detected in embryos undergoing abnormal versus normal development [9]
In this study, we seek to take advantage of RNA sequencing technology (RNA-seq) to identify transcriptomic changes associated with abnormal bovine early embryonic development, especially for transcriptional activities that have not been annotated before and differential alternative splicing that could not be detected by expression microarrays
Summary
Sequencing the bovine embryonic transcriptome To overcome the scarcity of RNA present in individual embryos, we linearly amplified RNA [10] from two pools of embryos: one comprised 20 embryos that properly completed embryonic development to blastocyst stage (hereafter referred to as ‘blastocysts’); the other consisted of 20 embryos that showed retarded morphology by the same developmental time point (hereafter referred to as ‘degeneratives’). Using Tophat, a total of 10,638 and 8,686 novel splice junctions supported by at least two fragments were discovered in blastocysts and degeneratives respectively, among which 8,315 were found in both samples (Table 1). The correlation between blastocysts and degeneratives was as high as 0.986 and relatively few genes (n = 47) were differentially expressed (Figure 1a, Additional File 1). A substantial fraction of novel TUs was supported by confident polyA-containing fragments, suggesting that the transcripts were potentially polyadenylated (Table 3). To test for differential expression between blastocysts and degeneratives for these novel TUs, raw fragment counts from novel TUs and annotated genes were combined, quantile normalized, and analyzed using the “DESeq” package. Three “alternative 5’ splice site” and 11 “alternative 3’ splice site” events showed nominal significance (p < 0.01), yet none of them retained significance after multiple testing correction
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