1. Comparative transcriptomics of fungi Group Nicotiana Daan van Vliet, Dou Hu, Joost de Jong, Krista Kokki

2. Research objective To study differences in gene expression in related fungi species Studies species: -Reference genome -RNA reads > 100 bp -Preferably: Paired-end -Related species (or at least: single-celled, eukaryotic) -Similar conditions

3. Data generation -Cleaning reads: FastQC (cut value: 25) -Mapping reads: TopHat -Assemble and quantify transcripts: Cufflinks -Extracting the transcripts: gffread

4. Data processing Extract top 100 expressed transcripts -Unix command pipeline within Perl (sort, head) Determining gc-content and length of transcripts -Perl subroutines

5. Data processing Determine intron length -Perl: length (transcript) – length of the exons (specified in Cufflinks output) Codon usage -Perl: loop to analyse codon by codon (within each transcript) -Count frequencies of codons, store in an array -Calculate codon usage index (such as the effective number of codons, N C ) Validation -Run scripts on small, example datasets

6. Statistical analysis and visualization Use R to determine and visualize correlation coefficients Expression level Species 1Species 2 Transcript length 0.25* -0.10* CG-content 0.00 0.02 Codon usage bias 0.30* -0.32*