the rna-‐seq analysis pipeline - alicia oshlack
DESCRIPTION
High-throughput sequencing of the transcriptome leads to the production of millions of short reads which need to be analysed in order to make biological inference. RNA-seq data are complex and the analysis involves a series of steps for which research is ongoing. However the choice of analysis methodology can have a major impact on the findings of an experiment. In this talk I will outline the major steps involved in analysing an RNA-seq dataset for the purpose of detecting differential expression. I will discuss and compare the available strategies for each stage of the pipeline. I will also point out several areas in RNA-seq data analysis which require further research in order for the full potential of the technology to be Realised.TRANSCRIPT
THE RNA-‐SEQ ANALYSIS PIPELINE
Alicia Oshlack Murdoch Childrens Research Ins5tute
Genes and transcripts
Gene
transcript
New genomic technologies are driving the science
Two ways to look at sequencing data
Sequence of(mapped) read • genome sequencing • variant detec5on • Muta5on detec5on • genomic rearrangements • Bisulfite-‐seq (methyla5on) • RNA edi5ng etc.
Posi5on of mapped read • RNA-‐seq • ChIP-‐seq • MeDIP-‐seq for DNA methyla5on etc.
4
Two ways to look at RNA-‐seq data
Sequence of(mapped) read • Assembly • Determining genes/transcripts
Posi5on of mapped read • Expression levels • Differen5al expression
5
Benefits and opportuni5es of RNA-‐seq
• All transcripts are sequenced not just ones for which probes are designed (cf microarrays)
• Annota5on of new exons, transcribed regions, genes or non-‐coding RNAs
• Whole transcriptome sequencing – The ability to look at alterna5ve splicing – Allele specific expression – RNA edi5ng
This talk
• Analysis of RNA-‐seq data for the purpose of determining differen5al expression
• How much are expression levels changing between samples?
RNA-‐seq
Pepke et al, Nature Methods, 2009
Raw data
• Short sequence reads • Quality scores
@SEQ_ID!GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT!+!!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65
RNA-‐seq analysis steps Raw sequence reads
Map onto genome
Summarize reads to transcripts
Sta5s5cal tes5ng: Determine differen5ally expressed genes
Systems biology
De novo assembly Annota5on based Genome guided assembly
Which transcriptome?
RNA-‐seq analysis steps Raw sequence reads
Map onto genome
Summarize reads to transcripts
Sta5s5cal tes5ng: Determine differen5ally expressed genes
Systems biology
De novo assembly Annota5on based Genome guided assembly
Which transcriptome?
Map reads to the genome
• Aligning millions of short sequencing onto the 3 billion bases of the human genome
• Accuracy vs speed • Many aligners available (BWA, Bow5e, STAR, Novoalign,…)
CDS CDS CDS CDS
CDS CDS CDS CDS
Gene
transcript
Sequencing transcripts not the genome
Splice site mapping • Build a junc5on library from all combina5ons of known exon boundaries
• Determine where splice junc5ons occur using the data itself -‐ unbiased by annota5on.
• Several so_ware packages to do this such as TopHat, SplitSeek, SpliceMap…
Gerber et al, Nat Methods, 2011
Which transcriptome to use?
Raw sequence reads
Map onto genome
Summarize reads to transcripts
Sta5s5cal tes5ng: Determine differen5ally expressed genes
Systems biology
RNA-‐seq analysis steps
De novo assembly Annota5on based Genome guided assembly Which transcriptome?
Op5on 1
• Use annota5on (known genes) – Works well for well studied organisms (human, mouse, arabidopsis, drosophila,…)
– only as good as your annota5on – No novel transcripts are analysed
Op5on 2: Genome guided transcript assembly
• Uses the loca5on and density of reads along the genome to assemble transcripts
• E.g. Cufflinks
• Can’t assemble across breaks in the genome – Cancer, poor genomes
Op5on 3: De novo transcriptome assembly
• Assemble transcripts from the data without using a reference genome
• “Harder” than genome assembly – Orders of magnitude varia5on in coverage – Con5gs are short – Alterna5ve isoforms/transcripts have overlapping sequences – *Very* computa5onally intensive
• So_ware includes – Oases (velvet) – TransAbyss – Trinity – …
Example: Annota5ng the chicken W chromosome
Z Z Z W
Male Female
Two hypotheses for mechanisms of avian sex determina5on: 1. Dominant ovary determining gene on W (cf mammals) 2. Dosage of Z-‐linked genes
There is an annotated chicken genome
• Chicken W chromosome is poorly assembled • Are genes on other chromosomes really on the W, in par5cular the random chromosome?
Chromosome Assembled Size (Mb)
Size inc. random (Mb)
Estimated Size (Mb)
Estimated Genes
(Ensembl) Z 69 70 80 796
W 0.24 0.89 18-54 46
Un_random 56 - - 1287
Experimental design
PCR Sexing
+12hour Blastoderms
Hand plate for PCR Sexing
RNA
Stage 26 paired gonads (day 4.5)
12 Female
RNA
16 Female gonads
Pooled Samples
12 Female
16 Female gonads
12 Male
12 Male
16 Male gonads
16 Male gonads
RNA-‐seq • Illumina HiSeq2000 • Paired-‐end 100bp • 4 lanes • >80million reads/sample
Defining the transcriptome
• Annota5on ~20,000 genes • Genome guided assembly (Cufflinks) ~45,000 genes
• De novo transcriptome assembly ~2.5 million transcripts (Abyss with filtering)!
A combined approach • Assemble cufflink genes using transcripts from our de novo assembly
Annota5on of the chicken W combined all three approaches
W/W_random ChromsomeUn_random ChromosomeAutosomes
Blastoderm CoverageGonads Coverage
Abyss TranscriptsCufflinks TranscriptsEnsembl Transcripts
1000 1500 2000 2500 3000 3500
base position
039
1
Coverage
Genome
EnsemblCufflinks
AbyssRASA1−W
1400 1600 1800 2000 2200
base position
012
6
Coverage
Genome
EnsemblCufflinks
AbyssST8SIA3−W
2000 2200 2400 2600 2800
base position
069
9
Coverage
Genome
EnsemblCufflinks
AbyssGOLPH3−W
0 500 1000 1500 2000 2500
base position
019
4
Coverage
Genome
EnsemblCufflinks
AbyssZSWIM6−W
0 200 400 600 800 1000 1200 1400
base position
082
Coverage
Genome
EnsemblCufflinks
AbyssNEDD4−like−W
Full list of W genes/transcripts for differen5al expression Ayers et al, 2013
Summariza5on
Take your “transcriptome” and add up the reads
Coding Sequence Exons Introns Splice Junc5ons
CDS CDS CDS CDS
§ Reads in exons § Exons + junc5ons § All reads start to end of transcript § De novo methods
CDS CDS CDS CDS
CDS CDS CDS CDS
CDS CDS CDS
Even when all transcripts are “known” summariza5on or expression quan5fica5on is difficult. How do you assign reads to transcripts?
Transcript 1
Transcript 2
CDS CDS CDS CDS Transcript 3
Summariza5on turns mapped reads into a table of counts
Tag ID! A1! A2! B1! B2!ENSG00000124208! 478! 619! 4830! 7165!ENSG00000182463! 27! 20! 48! 55!ENSG00000125835! 132! 200! 560! 408!ENSG00000125834! 42! 60! 131! 99!ENSG00000197818! 21! 29! 52! 44!ENSG00000125831! 0! 0! 0! 0!ENSG00000215443! 4! 4! 9! 7!ENSG00000222008! 30! 23! 0! 0!ENSG00000101444! 46! 63! 54! 53!ENSG00000101333! 2256! 2793! 2702! 2976!
…" … tens of thousands more tags …"
** very high dimensional data **
RNA-‐seq analysis steps Raw sequence reads
Map onto genome
Summarize reads to transcripts
Sta5s5cal tes5ng: Determine differen5ally expressed genes
Systems biology
De novo assembly Annota5on based Genome guided assembly
Which transcriptome?
Assessing differen5al expression (DE)
• Which genes are changing in their abundance between samples?
• Sta5s5cal tests for DE (edgeR)
Normaliza5on
Accoun5ng for/removing technical sources of varia5on
Library size = sum of each column
Tag ID! A1! A2! B1! B2!ENSG00000124208! 478! 619! 4830! 7165!ENSG00000182463! 27! 20! 48! 55!ENSG00000125835! 132! 200! 560! 408!ENSG00000125834! 42! 60! 131! 99!ENSG00000197818! 21! 29! 52! 44!ENSG00000125831! 0! 0! 0! 0!ENSG00000215443! 4! 4! 9! 7!ENSG00000222008! 30! 23! 0! 0!ENSG00000101444! 46! 63! 54! 53!ENSG00000101333! 2256! 2793! 2702! 2976!
…" … tens of thousands more tags …"
Simple thought experiment
• Two samples A and B, sequenced to the same depth (same library size)
• Every gene that is expressed in A, is expressed in B at the same level
• Say there are a small number of genes that are expressed uniquely to sample B, but they are quite highly expressed (lots of reads)
• Many genes within the common set will appear differen5ally expressed (B < A)
Another way to view it
• Hypothe5cal example: Sequence 6 libraries to the same depth, with varying levels of unique-‐to-‐sample expression
• Differences in observed counts among the common genes
Red=low, goldenyellow=high
Marioni et al. 2008 RNA-‐seq data
Distribu5on of log-‐ra5os of counts 0 counts are omived Red line = trimmed mean
trimmed mean
• 5 lanes of liver RNA, 5 lanes of kidney RNA • Compare two single kidney libraries (technical replicates), a_er adjus5ng for library size
.
(a)
log2(Kidney1 NK1) − log2(Kidney2 NK2)D
ensi
ty
-6 -4 -2 0 2 4 6
0.0
0.4
0.8
log2(Liver NL) - log2(Kidney NK)
Den
sity
-6 -4 -2 0 2 4 6
0.0
0.2
0.4(b)
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-20 -15 -10
-50
5
A = log2( Liver NL Kidney NK)
M=
log 2
(Liv
erN
L)-
log 2
(Kid
ney
NK)
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Housekeeping genesUnique to a sample
(c)
Same sample. Technical replicates
Different samples. Liver vs Kidney
Another way to view the data
.
(a)
log2(Kidney1 NK1) − log2(Kidney2 NK2)
Den
sity
-6 -4 -2 0 2 4 6
0.0
0.4
0.8
log2(Liver NL) - log2(Kidney NK)
Den
sity
-6 -4 -2 0 2 4 6
0.0
0.2
0.4(b)
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-20 -15 -10
-50
5
A = log2( Liver NL Kidney NK)
M=
log 2
(Liv
erN
L)-
log 2
(Kid
ney
NK)
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Housekeeping genesUnique to a sample
(c)
• Sequencing “real estate” is fixed.
• Underlying RNA composi5on can be very different – e.g. several liver-‐specific genes
• An adjustment at the analysis stage should be made
The adjustment to data analysis is a scaling constant
• Assump5on: core set of genes that do not change in expression.
• Pick a reference sample, compute TMM rela5ve to reference • TMM (Trimmed Mean of M values) M=log ra5o
• Adjustment to sta5s5cal analysis: – Use addi5onal offset (GLM)
Robinson & Oshlack, Genome Biology, 2010
Sta5s5cal tes5ng for DE
• For EACH GENE, is the mean expression level for the gene under one condi5on significantly different from the mean expression level under a different condi5on?
Tag ID! A1! A2! B1! B2!ENSG00000124208! 478! 619! 4830! 7165!ENSG00000182463! 27! 20! 48! 55!ENSG00000125835! 132! 200! 560! 408!ENSG00000125834! 42! 60! 131! 99!
…" … tens of thousands more tags …"
39
DE analysis happens in R • Several packages currently available for DE analysis of RNA-‐seq data in R: – baySeq, – DEGSeq, – DESeq – edgeR – NBPSeq (+ more methods with less refined code, e.g. TSPM)
• edgeR developed at WEHI, so this is my favourite • edgeR is only package so far with full GLM capabili5es
• Great support on Bioconductor mailing list!
41
Counts from a Single RNA Sample Can be Modeled with Poisson Distribu5on
RNA sample
M = total number of reads ≈ 20 million (for example) λg = true propor5on of gene g yg = number of reads for gene g
Read 1 Read 2 Read 3 Read 4 Read 5 Read 6 …
Short reads
Map reads to genome
Davis McCarthy
42
Counts from a Single RNA Sample Can be Modeled with Poisson Distribu5on
RNA sample
M = total number of reads ≈ 20 million λg = true propor5on of gene g yg = number of reads for gene g
Large M, small λi à yg approximately Poisson, μg = Mλg
Read 1 Read 2 Read 3 Read 4 Read 5 Read 6 …
Short reads
Map reads to genome
What we’re interested in
What we observe
Davis McCarthy
43
A Small RNA-‐Seq Experiment (Tech Reps)
Condi5on A Condi5on B
λg1 λg2 λg3 λg4
yg1 yg2 yg3 yg4
Genes g = 1, …, 30k
M1
M2 M3 M4
E(ygi) = Mi λgi Reads Mi ≈ 20 million
True Technical Reps Show Poisson Varia5on for Each Gene
Data: Marioni et al., Genome Res, 2008 44
Davis McCarthy
45
A Small RNA-‐Seq Experiment (Biological Reps)
Condi5on A Condi5on B
λg1 λg2 λg3 λg4
yg1 yg2 yg3 yg4
Genes g = 1, …, 30k
M1
M2 M3 M4
E(ygi) = Mi λgi Reads Mi ≈ 20 million
Davis McCarthy
Biological Replicate Data shows Quadra5c Mean-‐Variance Rela5onship
(development cycle of slime mould, 2 samples at hr00, & 2 at hr04)
binned variance, sample variance
Data: Parikh et al, Genome Biology, 2010
46 Davis McCarthy
Need to account for biological varia5on
• Technical replicate counts for a gene vary according to a Poisson law, i.e. sequencing varia5on is Poisson
• Biological CV (BCV) is the coefficient of varia5on with which the (unknown) true abundance of the gene varies between RNA samples.
• edgeR models a quadra5c mean-‐variance rela5onship var(ygi)= μgi + φgμgi
2 with φg=Biological CV2
Robinson et al, 2010
Output of analysis
• A list of genes with p-‐values for tes5ng differen5al expression between samples
• A ranked list of genes
RNA-‐seq analysis steps Raw sequence reads
Map onto genome
Summarize reads to transcripts
Sta5s5cal tes5ng: Determine differen5ally expressed genes
Systems biology
De novo assembly Annota5on based Genome guided assembly
Which transcriptome?
RNA-‐seq analysis steps Raw sequence reads
Map onto genome
Summarize reads to transcripts
Sta5s5cal tes5ng: Determine differen5ally expressed genes
Systems biology
De novo assembly Annota5on based Genome guided assembly
Which transcriptome?
Learn something!
The future
• Effec5vely dealing with no genome or poor quality genomes (our new method, Corset)
• Integra5ng RNA-‐seq data with other genomics data
• Analysis methodology is cri5cal and s5ll developing for specific purposes
• Opportuni5es to use this data in new and imagina5ve ways – requires new analysis methodology
Acknowledgements
• Mark Robinson (Uni Zurich) • Mavhew Young (Cambridge) • Nadia Davidson (MCRI) • Mavhew Wakefield (WEHI) • Gordon Smyth (WEHI) • Davis McCarthy (Oxford)