Epsilon Archive for Student Projects

Abstract

The pig is besides an important livestock species also a model organism for human biomedical research. Knowledge of the porcine genome is essential for improving product quality, animal welfare and also the biomedical research. This is accomplished through investigating the transcribed regions of the genome by collecting, sequencing and analyzing transcribed sequences (mRNA) converted into a complimentary DNA (cDNA) providing a complete sets of expressed genes. Therefore, the objective of this study was to sequence and analyze 10,000 porcine normalized full-length cDNA clones. Total RNA was extracted from 11 tissues of a fetal clone of pig and a normalized full-length cDNA library was constructed by a commercial company. The cDNA clones were cultured in 384-well plates and sequenced using Sanger sequencing method. The sequence similarity search was performed using Basic Local Alignment Search Tool (BLAST) against the porcine genome, porcine cDNA, human cDNA and mouse cDNA databases. Combining sequences from this study and the dataset generated earlier, a total of 13,989 sequences of at least 50 bps or more were generated from an overall of 19,968 cDNA clones processed. From the overall clone sequences, a total of 12,220 sequences provided hit in one or more of the pig, human or mouse databases. Blasting against the pig genome provided larger hits of 10,857. On the other hand, the pig cDNA database has provided total hits of 6,597. The human and mouse cDNA provided a total hits of 4,786 and 2,801, respectively, that enable comparative analyses to identify the homologous pig genes. Only 52 sequences have the same start-site with their respective pig transcripts and the majority of sequences shown variation. A total of 3,164 genes were identified from the library. A large-scale collection and characterization of the normalized cDNA library using direct sequencing on 384-well plates provides a valuable tool for understanding and investigation of the pig genome.