Advanced RNA-Seq course Introduction...2011/08/25 · Top genes SAGE and CAGE CAGE gene Ratio...
Transcript of Advanced RNA-Seq course Introduction...2011/08/25 · Top genes SAGE and CAGE CAGE gene Ratio...
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Advanced RNA-Seq course
Introduction
Peter-Bram ’t Hoen
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Expression profiling
• DNA mRNA protein
• Comprehensive RNA profiling possible: determine the abundance of all mRNA molecules in a cell / tissue
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Expression profiling: applications
• Qualitative: which part of the genome is expressed, in which cells, which mRNA isoforms
• Quantitative: compare across conditions, understand biological processes / mechanisms
• Tumor vs. Normal tissue
• Knock-out vs. wild-type mouse
• Changing nutrient conditions in yeast
• Etc.
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Transcriptome analysis
• Genome-wide expression profiling• Serial Analysis of Gene Expression (SAGE)
• Expression Microarrays
• Digital Gene Expression (DeepSAGE)
• Shotgun RNA sequencing
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Expression microarray• Relative abundance
• Limited by content
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Serial analysis of gene expression (SAGE)• Sequence and count short tags representative for a transcript
• Absolute abundance of transcript
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NGS-based sequencing vs. microarray
• RNA-seq• Counting
• Absolute abundance of transcript
• All transcripts present
• Expression microarray• Recording hybridization signal to complementary probe
• Relative abundance
• Cross-hybridization possible
• Content limited
1 2 3
4 5 6
7 8 9
T G C T A C G A T …
T T T T T T T G T …
111 222 33
444 555 666
777 888 999
T G C T A C G A T …
T T T T T T T G T …
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Absolute transcript abundance
• Dynamic range dependent on sequencing method, sequencing depth and cell type
• Millions required • NOT Roche 454
• BUT Illumina, SOLiD, Helicos
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Deep sequencing-based expression profiling
• Tag-based: one read per transcript• DeepSAGE most 3’ CATG
• DeepCAGE 5’-end
• PolyA -> ultimate 3’-end
• RNA-Seq: multiple reads per transcript• Whole mRNA sequencing after fragmentation
• miRNA (short RNA) sequencing
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DeepSAGE – sample preparation
PCR enrichment and gel purification (~85bp)
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Example gene: Gapd
14542
12555
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Example gene: alternative polyadenylation
97
99
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CAGE (Cap analysis of gene expression)
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Example CAGE
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More new transcription start sites (CAGE)
Better annotation of promoter regions
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General RNA-seq sample prep
1. Isolation of polyA+ mRNA with oligo-dT
2. Fragmentation by heating for 8 min at 94°C
3. Random-primed first and second strand cDNAsynthesis
4. End repair
5. Fragmentation
6. Adenylation of 3’-ends
7. Ligation of adapters (containing barcodes)
8. PCR amplification (15 cycles)
9. Clean-up
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Example RNA-Seq
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Alternative splicing
Mortazavi et al. Nature Methods 5, 621 - 628 (2008)
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Strand-specific random-primed sequencing
Cloonan, Nature Methods 2008
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Ovation: not so random-primed
• No polyA+ selection
• No fragmentation
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Helicos single molecule sequencing
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Example RNA-Seq (Helicos)ADAMTS8
ADAMTS15
NOV
Peter Henneman
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Example polyA profiling on Helicos
Eleonora de Klerk
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Example polyA profiling
Eleonora de Klerk
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miRNA sequencing
• SOLiD small RNA (whole transcriptome) seq kit
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Helicos direct RNA sequencing
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Analysis steps - Introduction
1. Alignment to genome (transcriptome)
2. Remapping of unaligned reads
3. (Determining transcript isoform structures)
4. Quantifying transcript abundanceRPKM: reads per kilobase per million reads
FPKM: fragments per kilobase per million reads
5. Statistical testing for differential expression
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Comparison to microarrays
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Illumina features: Excellent reproducibility
Raw data Square root-transformed and scaled data
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Excellent reproducibility between labs
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Analysis of replicate samples
• Pooling: small contaminations can have large effect on outcome
• Technical replicates: not really necessary when sufficient sequencing depth is reached
• Biological replicates important for determination of biological variation
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Power comparison
Van Iterson, BMC Genomics, 2009
Power
Number of samples
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Power comparison (2)Intensity range
Power
Number of samples
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CAGE vs. SAGE
C2C12myoblast
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Correlation CAGE vs. SAGE (gene level)
Logratio differentiated vs. proliferating
32341702Unchanged
21602144Differentially expressed (*)
UnchangedDifferentially expressed (*)
Differentially expressed genes
SAGE
CA
GE
* Bayesian error rate < 0.05Vencio et al. Bioinformatics 5: 119 (2004)
SAGE
CA
GE
Only detected with CAGE: 1169Only detected with SAGE: 1747
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Top genes SAGE and CAGECAGE gene Ratio Microarray SAGE gene Ratio Microarray
Hfe2 4,073 NA RP23-36P22.5 576 NA
Myom3 1,624 NA Neb 525 NA
Lmod2 1,305 NA Mylpf 504 Yes
Myh7 1,124 Yes Ttn 380 NA
Mb 908 Yes Myh3 368 Yes
RP23-36P22.5
735 NA Xirp1 306 Yes
Pygm 717 Yes 1110002H13Rik
263 NA
Myl4 614 Yes Tnnc1 232 Yes
Synpo21 595 NA Cav3 150 Yes
Myh1 561 Yes Cbfa2t3 133 Yes
…… ……
13 out of 30 not found by microarray 10 out of 30 not found by microarray
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Most significant pathwaysCAGE GO SAGE GO Microarray GO
Regulation of striated muscle contraction
Regulation of muscle contraction
Cycline-dependent protein kinase inhibitor activity
Cardiac muscle contraction Cardiac muscle contraction Myogenesis
Myogenesis Myogenesis Skeletal muscle development
Regulation of muscle contraction
Regulation of striated muscle contraction
Myoblast differentiation
Skeletal muscle development
Skeletal muscle development
6-phosphofructokinase activity
Muscle development Myofibril assembly Muscle development
Striated muscle contraction Muscle development Muscle cell differentiation
Myoblast differentiation Myoblast fusion Tumor suppressor activity
Muscle cell differentiation Striated muscle contraction Myofibril assembly
Sarcomere organization Muscle cell differentiation Heart development
10/10 muscle related 10/10 muscle related 7/10 muscle related
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Conclusions
• Next generation sequencing provides higher power, sensitivity and reproducibility than expression microarrays
• Deep sequencing offers more than microarrays• Alternative transcription start site usage
• Alternative splicing
• Alternative polyadenylation
• Allele-specific expression
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AcknowledgementsYavuz AriyurekHenk BuermansTassos MastrokoliasMatt HestandJohan den DunnenGertjan van Ommen
DNAFORM
Andreas KlinghoffMatthias ScherfThomas Werner
Matthias HarbersMakoto Suzuki
Wilbert van Workum