Base calling was carried out using the Illumina GA pipeline v1. 4. For each library, the filtered reads from the Illumina GA II pipeline were mapped using Tophat, a splice site aware short read mapper that works in conjunction with Bowtie short read aligner. Reads were deposited in the NCBI Short Read Archive. The minimum and maximum intron sizes selleckchem Ponatinib were 5 bp and 15 kbp, respectively, for each Tophat run. The final annota tion GFF3 file was provided to Tophat and expression values were calculated using reads per kilobase of exon model per million mapped reads. The minimum RPKM for all eight conditions was 0, the median RPKM ranged from 5 to 8, while the maximum RPKM ranged from 10,182 in the YEP Plich library to 32,041 Inhibitors,Modulators,Libraries from the 35 C temperature treatment. Inhibitors,Modulators,Libraries Using a RPKM value of 2.
5 as a cutoff for expression, loci with differential expression in treatment versus control were identified. The fold changes were calculated for loci with RPKM 2. 5 in both treatment and control samples. Loci with con trol values 2. 5 RPKM but with expression in treatment conditions were flagged as Inhibitors,Modulators,Libraries U as a true ratio could not be calculated. Loci with treatment values 2. 5 RPKM Inhibitors,Modulators,Libraries but with expression in control conditions were flagged as D as a true ratio could not be calculated. Loci with RPKM 2. 5 in both treatment and control samples were flagged as N. Identification of secreted proteins and effector families The secretome of P. ultimum was identified using Sig nalP V2. 0 program following the PexFinder algorithm as described previously.
In addition, sequences that were predicted to contain transmembrane domains or organelle targeting signals were omitted from the secretome. Each sequence in the secretome was searched Inhibitors,Modulators,Libraries against two Darwin databases that were compiled from 50 eukaryote whole proteomes from major phylogenetic branches make it clear using BLASTP with an E value cutoff of 1 10 3. One database contained sequences only from Fungi and the other contained the sequences from other organisms excluding the Fungi and oomycetes. Protein sequences of the secre tome were clustered into families along with their related non secretory proteins by using the TRIBE MCL algorithm using BLASTP with an E value cutoff of 1 10 10. Each family was named according to the existing annotation of the member sequences. Families and singletons were searched against Pfam A release 24. 0 using the HMMER3 beta 3 hmmsearch with trusted cutoffs to detect any transposable ele ment related proteins that may have been missed in the repeat masking process. Families or singletons where at least 50% of the members matched transpo son associated Pfam domains were manually curated to identify and exclude true transposon related sequences from the secretome. For the analysis of genome organization, P.