Transcriptome analysis Edouard Severing

Overview Introduction: Transcriptome complexity Transcriptome reconstruction Without a genome With a genome Transcript abundances Differential expression Transcript abundances models (Maximum likelihood)

Gene-expression/Phenotypes What are the gene expression differences that underly these phenotypic differences? Gene expression measured by assessing the abundance of mRNA molecules

Transcriptome vs. genome Initial assumption N mRNA Molecules N Proteins N Protein coding genes Assumption is based on studies that were performed on bacterial systems

Complexity and gene count 20.000 genes 25.000 genes

Transcriptome vs. genes in eukaryotes Current view X N mRNA Molecules ? N Proteins What happens here ? N Protein coding genes

Splicing Splicing 5’- -3’ 5’- -3’ 5’- -3’ Pre-mRNA Exon Intron Exon Gene

Alternative splicing II (Alternative splicing) Pre-mRNA 5’- -3’ 5’- -3’ Splicing 5’- -3’ 5’- -3’ Splicing

Complexity and AS 90% genes have AS 42% genes have AS The average number of transcripts produced by human genes is also higher than the average number of transcripts produced by plant genes

Extremes Dscam gene produces over 35,000 transcripts

AS type difference Humans Plants In humans exon skipping is most frequent AS event type In plants intron retention are the most common AS event type Humans Exon skipping Plants Intron retention

RNA editing (Base modification) Primary transcript (Predicted sequence) C U C 5’- A G U - 3’ A RNA-Editing After editing (Observed sequence) A C U U 5’- A G U - 3’ A Difficulty: Distinguish genuine RNA-editing from sequencing errors

Translation or decay A large fraction (>30%) of transcripts of protein coding genes are degraded by the nonsense-mediated decay (NMD) pathway. The position of the stop codon is used to predict whether a transcript is likely to be degraded by the NMD pathway

NMD target prediction Pre-mRNA 5’- -3’ Exon/Exon junctions mRNA 5’- Open reading frame M 5’- -3’ Stop d Transcripts containing a Stop codon more than 55 nt upstream of the last exon/exon junction are predicted to be targets for the NMD-pathway.

Remember The number of unique mRNA molecules is much larger than the number of genes. A large fraction of the mRNA molecules is degraded by the NMD pathway. NMD provides a means to regulate gene-expression at the post-transcriptional level

Transcriptome analysis. Reconstruction of the expressed transcripts given the sequencing data (Fragmented). Without a reference genome Trinity, TransABySS and Velvet With a reference genome Cufflinks, Scripture Determining the relative abundances of the predicted transcripts (cufflinks) Differential analysis (cufflinks) Gene-expression Alternative splicing

Without genome I

Without genome II

With a genome (Spliced alignment) 5’- -3’ mRNA

With a genome

With Genome II

Assignment Transcriptome reconstruction Mapping of reads to the genome using tophat Reconstruction of the transcriptome using cufflinks Blast analysis of the assembly result

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Details ssh –X <yourlogin>@137.224.100.212 cd /mnt/geninf15/work/bif_course_2012 assignments are in assignment.txt

Estimating Expression levels Would be easy if only full length transcripts were recovered. However, we have transcript fragments. Simply counting the number of reads mapping to a gene or transcript is not good enough (Normalization is needed) The number of fragments that can be produced from a transcript not only depends its abundance but also its length.

Expression levels FPKM is analogous to RPKM One fragment One read

Back to gene level expression (I)

Back to gene level expression (II)

Differential expression analysis A genes is differentially expressed under two conditions if its expression difference is statistically significant. Larger that you would expect based random natural variation - In order to estimate the variance it is important to have experimental replicates . (Variation between biological replicates is larger than that between technical replicates).

Expression assignment Estimate the expression levels of predicted transcripts / genes in Arabidopsis roots and flower buds. (Cufflinks) Differential expression analysis of transcript abundances in Arabidopsis roots and flower buds (Cuffdiff)