v10 Laura Biggins Interpretation Library Contamination Biological Interpretation Technical Tracking Interpreting results QC and visualisation still important Easy to draw wrong conclusions from the data ID: 542446
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Slide1
Failures in interpretation of sequencing results
v1.0Laura BigginsSlide2
InterpretationSlide3
Library
Contamination
Biological
Interpretation
Technical
Tracking
Interpreting results
QC and visualisation still important
Easy to draw wrong conclusions from the data Slide4
Interpretation exercise
Interpretation
RNA-
seq
dataSlide5
Interpretation
Sample 1 genome viewSlide6
Interpretation
Sample 2 genome viewSlide7
Interpretation
Genes
upregulated
in sample 1Slide8
Interpretation
Genes
downregulated
in sample 1Slide9
Interpretation
Interpretation exerciseSlide10
Functional Enrichment Analysis
Gene ontology analysisPathways analysisAny set of predefined functional categories with genes assigned to the categories
Enrichment test – gene list
vs
background
Useful and powerful but easy to produce false positives
InterpretationSlide11
Location bias
GO analysis of all genes on chromosome (mouse)
Interpretation
Michael Reik
Chr
Category
BH
adj
p-value
Chr 1
GO:0050662~coenzyme binding
1.61E-02
Chr 2
GO:0007608~sensory perception of smell
1.54E-64
Chr 3
GO:0005509~calcium ion binding
2.32E-01
Chr 4
GO:0009615~response to virus
2.58E-07
Chr 5
GO:0001730~2'-5'-oligoadenylate synthetase activity
2.44E-04
Chr 6
GO:0005529~sugar binding
1.54E-25
Chr 7
GO:0004984~olfactory receptor activity
1.08E-30
Chr 8
GO:0042742~defense response to bacterium
2.36E-16
Chr 9
GO:0007608~sensory perception of smell
2.68E-12
Chr 10
GO:0008227~amine receptor activity
3.24E-05
Chr 11
GO:0045111~intermediate filament cytoskeleton
1.79E-23
Chr 12
GO:0034097~response to cytokine stimulus
2.59E-04
Chr 13
GO:0000786~nucleosome
2.47E-17
Chr 14
GO:0004522~pancreatic ribonuclease activity
2.09E-22
Chr 15
GO:0045095~keratin filament
1.28E-17
Chr 16
GO:0004869~cysteine-type endopeptidase inhibitor activity
8.62E-08
Chr 17
GO:0042611~MHC protein complex
5.39E-19
Chr 18
GO:0007156~homophilic cell adhesion
1.01E-26
Chr 19
GO:0005506~iron ion binding
2.12E-07
Chr X
GO:0045449~regulation of transcription
6.88E-04Slide12
Mapping to genome
multi-mappingGO enrichment for “differentially expressed” genes
- ribosomal categories (p < 1E-20)
- histones & chromatin assembly (p < 1E-7)
Interpretation
Michael ReikSlide13
GC content
InterpretationSlide14
GC content
Interpretation
GOrilla
analysis using mouse genes with GC content
> 60% (~170
genes)Slide15
GC content
Interpretation
GOrilla
analysis using mouse genes with GC content < 35% (~200 genes)Slide16
Public RNA-seq
Public RNA-seq data from a range of mouse tissues Called differentially expressed genes between replicates within datasets
Gene lists enriched in functional categories:
Ribosome
Extracellular
Secreted
Glycoprotein
Myofibril, cytoskeleton
InterpretationSlide17
GO analysis of genes that appeared
in > 5 datasets
Extracellular
, glycoprotein categories
absent – large, diverse categories
Interpretation
Public RNA-
seqSlide18
Membrane associated transcripts
Re-sequencing library = same result
Remaking library from tissue = changes gone
InterpretationSlide19
Sfi1 – spindle associated
transcript
Interpretation
Misbehaving genesSlide20
Misbehaving genes
Titin, USH2A (big!)Mucin, Mid1, Sfi1 (duplication events)Olfactory receptors (big families)Poorly annotated (RIKEN, EST, Gm123,RP11 etc
)
InterpretationSlide21
Power
Interpretation
RNA-seq
Gene/transcript length
Expression level
Bisulphite-seq
CpG
content
Slide22
Confounding factors
Interpretation