Variance stabilized data obtained with DESeq was used to generate the heatmaps of differentially expressed Ruxolitinib genes. To study the biological significance of differentially expressed genes, gene ontology based enrichment tests were conducted using a web based tool GOMiner. For this analysis Arabidopsis homologs of transcripts were obtained by BLAST searching the Arabidopsis protein database using blastx. BLAST search was run with the parameters of maximum high scoring segment pairs of 100, expect value for matches of 10 and the de fault matrix of BLOSUM62. To identify Arabidopsis homologs for gene models predicted from reference guided transcriptome mapping, gene sequences were extracted from the Eucalyptus reference genome se quence using gene coordinates from the gene annotation file generated using the Cufflinks package.
The extracted Inhibitors,Modulators,Libraries gene sequences were BLAST searched with the Arabidopsis protein database. The identified Arabidopsis homologs were used in GO enrichment tests. Identification of Inhibitors,Modulators,Libraries SNPs To study allelic expression SNPs from ten seedlings be fore the treatment and the same ten seedlings after treatment were analysed. The BAM files generated from TopHat analysis were used for detecting SNPs. The BAM files were used in SAMTools to produce pileup files containing SNP information. Pileup files gen erated from SAMtools were analysed with Inhibitors,Modulators,Libraries VarScan soft ware to count the reads mapping to each allele of a variant and to estimate the allele frequencies. The fol lowing options were used in VarScan to detect the SNPs.
A minimum coverage of 8 reads mapping to variant sites, minimum base phred quality Inhibitors,Modulators,Libraries of 20 and a P value of 0. 05 were used for SNP calling. Reads from the three control treatment Inhibitors,Modulators,Libraries libraries and reads from the three stress treatment libraries were combined for detect ing SNPs. Read counts of variant alleles from control and stress treatments were used in testing for differential allelic expression using chi squared tests. Only consistent SNPs i. e. SNPs with the same alleles from both control and stress treatment were used in the differential allelic expression analysis. SNPs with a cover age of less than 20 reads in both the treatments were not used. Significance of the differential allelic expres sion was based on FDR. The BEDTools package was used to identify gene features as well as E. grandis genes overlapping SNPs.
Identification of genes under selection To study the selection patterns of genes we have esti mated the proportion of nonsynonymous to synonymous substitutions. We used the PoPoolation package to identify and to annotate selleck chemicals Nutlin-3a SNP variants i. e. to determine if an SNP is nonsynonymous or synonymous This tool uses a pileup file generated from SAMTools and a gene annotation file of coding sequences to identify and to annotate the SNP variants.