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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
Protein coding
genes
N
mRNA
Molecules
N
Proteins
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
N
Protein coding
genes
XN
mRNA
Molecules
What happens here ?
?N
Proteins
Splicing
Pre-mRNA
5’-
5’-
-3’
Exon
Intron
Exon
Gene
Intron
Splicing
mRNA
5’-
Exon
Exon
Exon
-3’
Exon
-3’
Alternative splicing II
(Alternative splicing)
Pre-mRNA
5’-
-3’
-3’
5’-
5’-
-3’
Splicing
5’-
Splicing
-3’
5’-
-3’
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
In humans exon skipping is most frequent AS event type
In plants intron retention are the most common AS event
type
Humans
Plants
Exon skipping
Intron retention
RNA editing
(Base modification)
Primary transcript
(Predicted sequence)
5’- A
C
U
A
C
G
A
U - 3’
RNA-Editing
After editing
(Observed sequence)
5’- A
C
U
A
U
G
A
U - 3’
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’-
mRNA
-3’
5’-
-3’
Exon/Exon junctions
M
Open reading frame
Stop
5’-
-3’
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)
Genome
-3’
5’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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Change password
• ssh <yourlogin>@137.224.100.201
• passwd
– Enter your password
– Change it to new password
– Type new password again
• Exit
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
Number of reads mapped to a region
RPKM  10 x
Total reads x region length
9
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)