RNA-Seq Analysis

Simon Andrewssimon.andrews@babraham.ac.uk

@simon_andrewsv2.3

RNA-Seq Libraries

rRNA depleted mRNA

Fragment

Random prime + RT

2nd strand synthesis (+ U)

A-tailing

Adapter Ligation

(U strand degradation)

Sequencing

NNNN

u u u u

u u u u AA

u u u u AAT

T

A T

RNA-Seq Analysis

QC (Trimming) Mapping

Mapped QCQuantitationStatistical Analysis

QC: Raw Data• Sequence call quality

QC: Raw Data• Sequence bias

QC: Raw Data• Duplication level

Mapping

Exon 1 Exon 2 Exon 3 Genome

Simple mapping within exons

Mapping between exons

Spliced mapping

Can simplify by aligning first to a transcriptome and then translate back to genomic coordinates. Can map unmatched reads to the whole genome.

Spliced Mapping Software

• Tophat (http://tophat.cbcb.umd.edu/)

• Star (http://code.google.com/p/rna-star/)

TopHat pipelineReference FastQ files Indexed Genome

Reference GTF Models Indexed Transcriptome

Reads Maps to transcriptome? Translate coords and report

Maps to genome?

Split map to genome Build consensus and report

Report

Yes

Yes

Yes

Discard

No

No

No

Post Mapping QC

• Mapping statistics• Proportion of reads which are in transcripts• Proportion of reads in transcripts in exons• Strand specificity• Consistency of coverage

SeqMonk (RNA-Seq QC plot)RNASeqQC (easiest through GenePattern)

SeqMonk Mapping QC (good)

SeqMonk Mapping QC (bad)

Quantitation

Exon 1 Exon 2 Exon 3

Exon 1 Exon 3

Splice form 1

Splice form 2

Definitely splice form 1

Definitely splice form 2

Ambiguous

Options for handling splice variants

• Ignore them – combine exons and analyse at gene level– Simple, powerful, inaccurate in some cases– DE-Seq, SeqMonk

• Assign ambiguous reads based on unique ones – quantitate transcripts and optionally merge to gene level– Potentially cleaner more powerful signal– High degree of uncertainty– Cufflinks, bitSeq, RSEM

Read counting

• Simple (exon or transcript)– HTSeq (htseq-count)– BEDTools (multicov)– featureCounts– SeqMonk (graphical)

• Complex (re-assignment)– Cufflinks, bitSeq, RSEM

Count Corrections

• Size of library

• Length of transcript

• Other factors

RPKM / FPKM / TPM• RPKM (Reads per kilobase of transcript per million reads of library)

– Corrects for total library coverage– Corrects for gene length– Comparable between different genes within the same dataset

• FPKM (Fragments per kilobase of transcript per million reads of library)

– Only relevant for paired end libraries– Pairs are not independent observations– RPKM/2

• TPM (transcripts per million)

– Normalises to transcript copies instead of reads– Corrects for cases where the average transcript length differs between samples

Normalisation

Normalisation

Filtering Genes

• Remove things which are uninteresting or shouldn’t be measured

• Reduces noise – easier to achieve significance– Non-coding (miRNA, snoRNA etc) in RNA-Seq– Known mis-spliced forms (exon skipping etc)– Mitochonidrial genes– X/Y chr genes in mixed sex populations– Unknown/Unannotated genes

Filtering Mouse mRNAs

Non coding

ESTs

Predicted genes

Good transcripts

Visualising Expression

• Comparing the same gene in different samples– Normalised log2 RPM values

• Comparing different genes in the same sample– Normalised log2 RPKM values

Linear Log2

Eef1a1

Actb

Lars2

Eef2

CD74

Differential Expression

• Microarrays traditionally used continuous statistical tests (t-test ANOVA etc)

• RNA-Seq differs in that it is count based data, so continuous tests fail at low counts

• Most differential tests use count based distribution tests, usually based on a negative binomial distribution

Negative binomial tests (DE-Seq)

• Are the counts we see for gene X in condition 1 consistent with those for gene X in condition 2?

• Initially modelled using simple Poisson distribution using mean expression as the only parameter

• Doesn’t model real data very well

Poisson vs Negative binomial

PoissonDESeq

Parameters• Size factors– Estimator of library sampling depth– More stable measure than total coverage– Based on median ratio between conditions

• Variance – required for NB distribution– Custom variance distribution fitted to real data– Insufficient observation to allow direct measure– Smooth distribution assumed to allow fitting

Dispersion shrinkage• Plot observed per gene dispersion

• Calculate average dispersion for genes with similar observation

• Individual dispersions regressed towards the mean. Weighted by– Distance from mean– Number of observations

• Points more than 2SD above the mean are not regressed

Other filters• Cook’s Distance – Identifies high variance– Effect on mean of removal of one replicate– For n<3 test not performed– For n = 3-6 failures are removed– For n>6 outliers removed to make trimmed mean– Disable with cooksCutoff=FALSE

• Hit count optimisation– Low intensity reads are removed– Limits multiple-testing to give max significant hits– Disable with independentFiltering=FALSE

Replicates

• Compared to arrays, RNA-Seq is a very clean technical measure of expression– Generally don’t run technical replicates

• Some statistics can be run on single replicates, but they can only tell you about technical noise (how likely is it that this change is due to a technical issue)

• Assessing biological variation requires biological replicates

Replicates• Traditional statistics require min 3x3

• DESeq can operate at 2x2, but this is minimum, not recommended

• True number of replicates required will depend on your biology and requirements

• 4x4 design is fairly common

• Always expect at least one sample to fail

• Randomise samples during sample prep

The problem of power…

• In a library Gene B is much better observed for the same copy number

• Power to detect DE is proportional to length

Gene A (1kb)

Gene B (8kb)

5x5 Replicates

5,000 out of 22,000 genes (23%) identified as DE using DESeq (p<0.05)

Intensity difference test

• Different approach to differential expression• Doesn’t aim to find every differentially

expressed gene• Conservative test• Guaranteed to never return large numbers of

hits

Assumptions

• Noise is related to observation level– Similar to DESeq

• Differences between conditions are either– A direct response to stimulus– Noise, either technical or biological

• Find points whose differences aren’t explained by general disruption

Method

Results

Exercises

• Look at raw QC• Mapping with tophat– Small test data

• Quantitation and visualisation with SeqMonk– Larger replicated data

• Differential expression with DESeq• Review in SeqMonk

Useful links• FastQC http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

• Tophat http://tophat.cbcb.umd.edu/• SeqMonk http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/

• Cufflinks http://cufflinks.cbcb.umd.edu/• DESeq http://www-huber.embl.de/users/anders/DESeq/

• Bioconductor http://www.bioconductor.org/• RSEM http://deweylab.biostat.wisc.edu/rsem/