Transcriptome Analysis of In Vitro- and In Vivo-Produced Preimplantation Porcine Embryos by 454 Pyrosequencing.

Abstract

Currently, our understanding of the molecular events during the development of porcine preimplantation embryos is limited. Increased knowledge in this area will contribute to our understanding of basic reproductive biology, will allow for identification of molecular markers related to embryonic quality, and will facilitate improvement to in vitro embryo production and culture conditions. The objective of this study was to identify transcripts related to the development of in vitro- (IVT) and in vivo (IVV)-produced porcine embryos using the 454-sequencing platform. Two normalized cDNA libraries were generated from pools of IVT and IVV embryos at different developmental stages (oocyte, 2-cell, 4-cell, 8-cell, morula and blastocyst). In order to facilitate the identification of rare transcripts, TRIMMER kit (Evrogen, Moscow, Russia) was used to decrease the prevalence of abundant transcripts and increase the efficiency of 454 sequencing for discovery of rare copies. A preliminary sequence analysis of the IVT and IVV cDNA libraries from a 1/8 plate run on the 454-Genome Sequencer FLX system (Roche) using the Titanium reagent kit yielded 94,628 and 82,582 ESTs, respectively. NextGENe (SoftGenetics LLC) software was used to identify expressed genes from a variety of porcine sequence databases as references. These reference sequence collections were downloaded from Ensembl: Sus_scrofa.Sscrofa9.56.cdna.abinitio.fa, Sus_scrofa.Sscrofa9.56.cdna.all.fa, and Sus_scrofa.Sscrofa9.56.dna.chromosome.*.fa; and from NCBI: rna.gbk. The collection of chromosomes were indexed using the ‘Build Index for WGA’ (whole genome alignment) tool of NextGENe. An average of 20% of expressed genes matched to the above references in ESTs obtained from both normalized cDNA libraries. Differential expression levels for IVT and IVV embryos were compared using the RPKM (reads per kilobase of exon model per million mapped reads) method and mapped to two databases (the NCBI rna.gbk genome and a combined Ensembl abinitio and cdna-all reference). Some commonly expressed genes were identified, and also, unique genes expressed either in IVT or IVV embryos. The remaining uncharacterized transcripts had significant matches against the EST porcine database. We further annotated the transcripts using the non-porcine mammalian protein database and Gene Ontology terms. This newly found EST resource will be valuable for embryo specific microarray development and for comparative mammalian genome analysis. This research was supported by the Natural Sciences and Engineering Research Council (NSERC) and is a part of the activities of the EmbryoGENE NSERC Strategic Research Network. (poster)