Comparative transcriptomics

of fungi

Group NicotianaDaan van Vliet, Dou Hu, Joost de Jong, Krista Kokki

Research objectiveTo 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

Data generation

- Cleaning reads: FastQC (cut value: 25) - Mapping reads: TopHat- Assemble and quantify transcripts:

Cufflinks- Extracting the transcripts: gffread

Data processing

Extract top 100 expressed transcripts- Unix command pipeline within Perl (sort,

head)

Determining gc-content and length of transcripts - Perl subroutines

Data processingDetermine 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, NC)

Validation- Run scripts on small, example datasets

Statistical analysis and visualization

Use R to determine and visualize correlation coefficients

Expression level

Species 1 Species 2

Transcript length 0.25* -0.10*CG-content 0.00 0.02Codon usage bias 0.30* -0.32*