Computational protocol: Identification of genes regulating ovary differentiation after pollination in hazel by comparative transcriptome analysis

Similar protocols

Protocol publication

[…] After removal of reads with adaptors, reads with more than 5% unknown bases (N), and low quality reads (defined as reads in which the proportion of bases with a quality score < 10 was greater than 20%), the remaining clean reads were stored in FASTQ format. Trinity (version: v2.0.6) [] was used to perform de novo assembly with clean reads from all 12 libraries with min_contig_length set to 150 and min_kmer_cov set to 3 and all other parameters set to default. Tgicl (version: v2.0.6) [] was used to cluster transcripts to Unigenes with repeat_stringency, minmatch, and minscore set to 0.95, 35, and 35, respectively, and all other parameters set to default. We used Blast [] to align unigenes to NT, NR, COG, KEGG and SwissProt to obtain annotations, Blast2GO (Version: v2.5.0; parameters: default) [] with NR annotation to obtain GO annotations, and InterProScan5 [] to obtain InterPro annotations. Clean reads were mapped to unigenes using Bowtie2 (version: v2.1.0) [] with options “q; phred64; sensitive; dpad, 0; gbar, 99999999; mp, 1, 1; np, 1; score-min L, 0, -0.1; I, 1; X, 1000; no-mixed; no-discordant; p, 1; k, 200”, and then, gene expression levels were calculated with RSEM []; all parameters were set to default. For different sample libraries (F-vs-S, S-vs-T, and T-vs-FO, in which the former was used as the control and the latter as the experimental group in each paired comparison), three pairs of DGE profiles were compared in order to determine changes in gene expression during ovary differentiation and ovule growth in hazel. Based on the unigene expression results, DEGs were identified with EBseq [] by setting the threshold of fold change as ≥2.00 and posterior probability of equivalent expression (PPEE) as ≤0.05. KEGG pathway enrichment analysis of DGE data was carried by a BLAST search of the KEGG database (http://www.kegg.jp/kegg/). Q ≤ 0.05 was used as the threshold for significant enrichment of DEG KEGG pathways. Hierarchical clustering analysis of DEGs was carried out using Multi Experiment Viewer (http://mev.tm4.org/#/welcome). […]

Pipeline specifications