Sequence analysis and transcript length estimation of a normalized full-length porcine cDNA library

Abstract: The Pig is one of the most important sources of animal protein and an essential animal model for human biomedical research due to its anatomical, physiological, biochemical and metabolic similarities with human being. Profound understanding of the pig genome helps to understand its biology in depth. To help this, availability of the pig genome sequence in the public databases facilitates research of wider spectrum. Generating Expressed sequence tags (EST) by partial sequencing normalized full-length cDNA libraries can improve the discovery of genes expressed in different tissues. cDNA libraries are also vital resources to elucidate alternative splicing pattern of genes and infer splice variants. The objective of this study was to pick and sequence the 5’end of 7,680 individual clones, identify clone sequences blasted against identical genes to determine the transcript size by Agarose-gel analysis and examining the presence of splice variants. Total RNA was isolated from eleven different tissues of an adult pregnant pig (113 days of gestation) and a normalized full-length cDNA library was constructed by a commercial company. Individual cDNA clones were cultured in 384-well plates and Sanger sequenced on an ABI3730 DNA analyser. The obtained sequence results were merged with results of two previous studies of the same cDNA library which resulted in 26,880 processed clones. In total 19,470 edited sequences were used for further analysis. Sequence similarity was searched using Basic Local Alignment Searching Tool (BLAST) against pig cDNA, human cDNA and mouse cDNA databases. A total of 12,461 sequences provided hit against the pig cDNA database and 8,300 sequences provided hit against the human cDNA database. 5,268 sequences provided hit against the mouse cDNA database. Moreover, the first 100 base pairs of the sequences were blasted against the newly released pig genome (build 10.2). In total of 12,222 sequences provided hit against the pig genome. Significant amount of clones provided database specific hits in either the Human or Mouse cDNA database which are important to retrieve homologous pig genes which are not mapped in the pig genome. A total of 7,074 non-redundant transcripts and 6,877 non-redundant genes were retrieved. PCR and Gel-electrophoresis protocols were optimized to determine insert the size of transcripts. Transcript length of 108 clones blasted against 10 different genes was determined and variation in size was obtained. The complete sequence of the 13 clones blasted to the same gene; the Swine Leucocyte Antigen (SLA-3) gene was obtained by sequencing with internal primers and variation in length and Exon-intron organization was confirmed. A comparison between the clone sequences and mRNA sequence stored in gene bank revealed higher degree of similarity and tissue specific transcripts were found. Large-scale screening and sequencing of cDNA libraries constructed from various tissues and development stages using the direct sequencing procedure can help to understand the pig genome. The procedure is also important to identify tissue specific splice variants.