Comparative transcriptomic analysis of fungi Group Nicotiana Daan van Vliet, Dou Hu, Joost de Jong, Krista Kokki

Research objective To study differences in gene expression in related fungus species Studies species: -Reference genome -RNA reads > 100 bp -Preferably: Paired-end -Related species -Similar conditions

Comparison Comparison between different species - Saccharomyces cerevisiae(yeast) - Komogataella pastoris(Pichia, yeast) - Aspergillus oryzae(fungus)

Methods – Data [Daan] RNA-seq: SRA Genome and annotation:Ensembl Fungi Read quality analysis performed with FastQC

Methods - Data processing Cleaning reads: SolexaQA Mapping reads: TopHat Assembly/Quantification: Cufflinks Optional replicate assembly: Cuffmerge Extracting transcript seqs: gffread Selection of top 100 genes: Linux

Methods – Gene properties PropertyExplanationTool (input datafile) ExpressionCount of mapped readsPerl script (fasta) LengthCount of base pairs of whole genePerl script (fasta) Intron lengthCount of base pairs within intronsPerl script (gtf) GC contentGC count/LengthPerl script (fasta) NcRatio: 20-61; 20 = one codon per amino acid; 61: random codon use CodonW (fasta) CG3sGC content of 3 RD synonymous codon position CodonW (fasta)

Methods – Interaction Top 100 genes were mapped to the interactome file and visualised through Cytoscape.

Hypothesis for yeast - Validation GC-content correlates positively with gene length. Negative correlation with gene length and degree of codon bias. Codon bias is more extreme in highly expressed genes. Genes with longer introns show higher bias in codon usage. The overall codon usage matches the known bias.

GO-terms and gene locations GOBPIDPvalueOddsRatioExpCountCountSizeTerm 1GO: E cytoplasmic translation 2GO: E primary metabolic process 3GO: E cellular component biogenesis at cellular level 4GO: E rRNA export from nucleus 5GO: E organelle assembly ChromosomeIIIIIIIVVVIVIIVIIIIXXXIXII XIVXVXVI Nro. of genes The top 5 most over-represented GO-terms for all the found genes The chromosomes the genes are found in.

Results – Correlations Gene expression vs. Gene length Saccharomyces cerevisiae Aspergillus oryzae Komogataella pastoris

Results – Correlations Gene expression vs. Intron length Saccharomyces cerevisiae Aspergillus oryzae Komogataella pastoris

Results – Correlations Gene expression vs. Effective Nr of codons Saccharomyces cerevisiae Aspergillus oryzae Komogataella pastoris

Results – Correlations Effective Nr of Codons vs. GC-cont. 3 rd pos. Saccharomyces cerevisiae Aspergillus oryzae Komogataella pastoris

Results – Correlations Gene length vs. Effective Nr of Codons Saccharomyces cerevisiae Aspergillus oryzae Komogataella pastoris

Results – Correlations Gene length vs. GC-content Saccharomyces cerevisiae Aspergillus oryzae Komogataella pastoris

Results – Correlations Gene length vs. Intron length Saccharomyces cerevisiae Aspergillus oryzae Komogataella pastoris

Results – Correlations Intron length vs. Nc Saccharomyces cerevisiae Aspergillus oryzae Komogataella pastoris

Results – Correlations Overall: -Within species: Few correlations between gene properties -Between species: Different patterns(?)

Cytoscape GO terms Top100 genes show different interactive network in GO terms

Results - First choice Yeast Interactome Project for S. cerevisiae high-throughput yeast two-hybrid (Y2H) provides high-quality binary interaction information. high-throughput Y2H dataset covering ~20% of all yeast binary interactions. This binary map is enriched for transient signalling interactions and inter-complex connections with a highly significant clustering between essential proteins.

Database choosing interactions from CCSB-YI1 1,809 interactions among 1,278 proteins

Second choice YeastNet v. 2 a probabilistic functional gene network of yeast genes, constructed from ~1.8 million expermental observations from DNA microarrays, physical protein interactions, genetic interactions, literature, and comparative genomics methods. In total, YeastNet v.2 covers 102,803 linkages among 5,483 yeast proteins a modified Bayesian integration of diverse data types, with each data type weighted according to how well it links genes that are known to share functions. (LLS)

Database choosing All the top 100 genes could find interactors in the Yeastnet v.2. We could find 9896 possibilities among 102,803 linkages

The end Questions?

Results – Correlations Gene expression vs. CG content Saccharomyces cerevisiae Aspergillus oryzae Komogataella pastoris