Computational protocol: Isolation and expression analysis of cDNAs that are associated with alternate bearing in Olea europaea L. cv. Ayvalık

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Protocol publication

[…] Insert (cDNA) sequences obtained from RefGen (Ankara, Turkey) were analysed using FinchTV v1.4 (Geospiza, Seattle, WA) and BioEdit [] for chromatogram quality and contig construction. Insert sequences confirmed for accuracy were blasted [] in BLASTn and BLASTx databases of NCBI - GenBank, and the homologous records from other plants were determined. When no significant hits from BLASTn and BLASTx databases were obtained, all other available databases were searched and the significant hits were recorded. Putative identities and functional annotation of the unique sequences obtained from the leaves (“on” and “off” years) and fruits were determined using the Blast2Go (B2G) software suite v2.3.1 with the default parameters (http://www.blast2go.com/b2ghome) []. The annotation process of assembled cDNA sequences (contigs) was mainly performed in three steps; (i) sequence similarity search of each individual ESTs was compared to the sequences in non-redundant database in GenBank by using the BLASTx algorithm with default settings, (ii) gene mapping and gene ontology (GO) categorization of unique sequences based on the BLASTx hits, and (iii) KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to decipher the biological function of unique sequences. After processing EST sequences such as sequence cleaning, vector masking and clustering, all ESTs were converted into fasta format. First, a set of olive EST sequences in fasta format were loaded into the B2G software and homology analysis against the NCBI nr protein database was conducted using a BLASTx algorithm (cutoff E-value of 1.0E-5 ) in order to identify the gene and protein names pertaining to the olive ESTs. Sequences having no homology (or BLASTx hits) to the protein database were further analyzed at the nucleotide level with BLASTx (cutoff E-value of 1.0E-5). The detailed BLAST results were automatically extracted and converted into excel tables including; sequence length, gene name, e-value, similarity, hit-length, align-length, GenBank and Uniprot accession numbers as well as Gene Ontology IDs belonging to each sequences (see Additional file ). During the annotation process, each olive EST was analyzed to address the functionality of newly identified genes using the KEGG automatic annotation server (KAAS-http://www.genome.ad.jp/tools/kaas/) which provides the functional annotation of genes based on sequence similarity comparisons against the genes within KEGG Genes database []. […]

Pipeline specifications

Software tools FinchTV, BioEdit, BLASTN, BLASTX, Blast2GO, KAAS
Application Sanger sequencing
Organisms Olea europaea
Chemicals Coenzyme A, Galactose, Glucose, Uridine Diphosphate