Computational protocol: Pancreatic PYY but not PPY expression is responsive to short-term nutritional state and the pancreas constitutes the major site of PYY mRNA expression in chickens

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Protocol publication

[…] The large amount of high-throughput sequencing information from the chicken (Gallus gallus) available in the sequence read archive (SRA) () was mined to derive a contiguous mRNA sequence for chicken PYY. Relevant experiments were identified by using appropriate search terms at the European Nucleotide Archive (). Data files were then probed for PYY mRNA sequence using the known chicken PYY peptide sequence (AAB24283.1) as a query in tblastn (NCBI). Short read sequences of interest were downloaded in FASTA format and read into GAP () for alignment. The process was iterative and as each new consensus was built the SRA files were re-interrogated using nucleotide BLAST until no further 3′ or 5′ extension was obtained. The final consensus sequence was analysed for open reading frames using ExPasy Translate () and likely signal peptide cleavage sites using SignalP ().For confirmation of the cDNA 5′ end in chicken, rapid amplification of cDNA ends (RACE) was completed using the Roche 2nd generation 5′/3′ RACE kit as per the manufacturer’s protocol and LightRUN Sanger sequencing (GATC Biotech) was employed to sequence the product. A similar data-mining process was followed for quail (Coturnix coturnix) PYY. This was only to increase confidence in inference to the evolving structures of vertebrate PYY and no further characterisation was carried out for quail PYY. The chicken PPY gene sequence was already definitively known (NM_204786.1) (). […]

Pipeline specifications

Software tools TBLASTN, SignalP
Databases ENA SRA ExPASy
Application Amino acid sequence alignment
Organisms Gallus gallus