RNA-Seq Work Flows
Martin Morgan, Sonali Arora
October 28, 2014
7-step work flow
1. Experimental design
Keep it simple
- Classical experimental designs
- Time series
- Without missing values, where possible
- Intended analysis must be feasbile – can the available samples and
hypothesis of interest be combined to formulate a testable
- Extent of replication determines nuance of biological question.
- No replication (1 sample per treatment): qualitative description
with limited statistical options.
- 3-5 replicates per treatment: designed experimental manipulation
with cell lines or other well-defined entities; 2-fold (?)
change in average expression between groups.
- 10-50 replicates per treatment: population studies, e.g., cancer
- 1000's of replicates: prospective studies, e.g., SNP discovery
- One resource: RNASeqPower
Avoid confounding experimental factors with other factors
- Common problems: samples from one treatment all on the same flow
cell; samples from treatment 1 processed first, treatment 2
processed second, etc.
Be aware of batch effects
- Leek et al., 2010, Nature Reviews Genetics 11
Leek & Story PLoS Genet 3(9):
- Scientific finding: pervasive batch effects
- Statistical insights: surrogate variable analysis: identify and
build surrogate variables; remove known batch effects
- Benefits: reduce dependence, stabilize error rate estimates, and
combat software / sva Bioconductor package
HapMap samples from one facility, ordered by date of processing.
Artifacts of your particular protocols
- Sequence contaminants
- Enrichment bias, e.g., non-uniform transcript representation.
- PCR artifacts – adapter contaminants, sequence-specific
amplification bias, …
Axes of variation
- Single- versus paired-end
- Length: 50-200nt
- Number of reads per sample
- ChIP-seq: short, single-end reads are usually sufficient
- RNA-seq, known genes: single- or paired-end reads
- RNA-seq, transcripts or novel variants: paired-end reads
- Copy number: single- or paired-end reads
- Structural variants: paired-end reads
- Variants: depth via longer, paired-end reads
- Microbiome: long paired-end reads (overlapping ends)
- de novo
- No reference genome; considerable sequencing and computational
- Established reference genome
- Splice-aware aligners
- Novel transcript discovery
- Established reference genome; reliable gene model
- Simple aligners
- Known gene / transcript expression
Splice-aware aligners (and Bioconductor wrappers)
(5a. Bowtie2 / tophat / Cufflinks / Cuffdiff)
- tophat uses Bowtie2 to perform basic single- and paired-end
alignments, then uses algorithms to place difficult-to-align reads
near to their well-aligned mates.
- Cufflinks (doi)
takes tophat output and estimate existing and novel transcript
How Cufflinks Works
- [Cuffdiff] assesses statistical significance of estimated
abundances between experimental groups
5. Reduction to 'count tables'
- Use known gene model to count aligned reads overlapping regions of
interest / gene models
- Gene model can be public (e.g., UCSC, NCBI, ENSEMBL) or ad hoc (gff file)
Step 6. Analysis
- Counts per se, rather than a summary (RPKM, FRPKM, …), are
relevant for analysis
- For a given gene, larger counts imply more information; RPKM etc.,
treat all estimates as equally informative.
- Comparison is across samples at each region of interest; all
samples have the same region of interest, so modulo library size
there is no need to correct for, e.g., gene length or mapability.
- Libraries differ in size (total counted reads per sample) for
un-interesting reasons; we need to account for differences in
library size in statistical analysis.
- Total number of counted reads per sample is not a good estimate of
library size. It is un-necessarily influenced by regions with large
counts, and can introduce bias and correlation across
genes. Instead, use a robust measure of library size that takes
account of skew in the distribution of counts (simplest: trimmed
geometric mean; more advanced / appropriate encountered in the lab).
- Library size (total number of counted reads) differs between
samples, and should be included as a statistical offset in
analysis of differential expression, rather than 'dividing by' the
library size early in an analysis.
Appropriate error model
- Count data is not distributed normally or as a Poisson process,
but rather as negative binomial.
- Result of a combination Poisson (shot' noise, i.e., within-sample
technical and sampling variation in read counts) with variation
between biological samples.
- A negative binomial model requires estimation of an additional
parameter ('dispersion'), which is estimated poorly in small
- Basic strategy is to moderate per-gene estimates with more robust
local estimates derived from genes with similar expression values (a
little more on borrowing information is provided below).
- Naively, a statistical test (e.g., t-test) could be applied to each
row of a counts table. However, we have relatively few samples
(10's) and very many comparisons (10,000's) so a naive approach is
likely to be very underpowered, resulting in a very high false
- A simple approach is perform fewer tests by removing regions that
could not possibly result in statistical significance, regardless of
hypothesis under consideration.
- Example: a region with 0 counts in all samples could not possibly be
significant regradless of hypothesis, so exclude from further
- Basic approaches: 'K over A'-style filter – require a minimum of A
(normalized) read counts in at least K samples. Variance filter,
e.g., IQR (inter-quartile range) provides a robust estimate of
variability; can be used to rank and discard least-varying regions.
- More nuanced approaches: edgeR vignette; work flow
- Why does low statistical power elevate false discovery rate?
- One way of developing intuition is to recognize a t-test (for
example) as a ratio of variances. The numerator is
treatment-specific, but the denominator is a measure of overall
- Variances are measured with uncertainty; over- or under-estimating
the denominator variance has an asymmetric effect on a t-statistic
or similar ratio, with an underestimate inflating the statistic
more dramatically than an overestimate deflates the statistic. Hence
elevated false discovery rate.
- Under the typical null hypothesis used in microarray or RNA-seq
experiments, each gene may respond differently to the treatment
(numerator variance) but the overall variability of a gene is
the same, at least for genes with similar average expression
- The strategy is to estimate the denominator variance as the
between-group variance for the gene, moderated by the average
between-group variance across all genes.
- This strategy exploits the fact that the same experimental design
has been applied to all genes assayed, and is effective at
moderating false discovery rate.
Step 7. Comprehension
Placing differentially expressed regions in context
- Gene names associated with genomic ranges
- Gene set enrichment and similar analysis
- Proximity to regulatory marks
Integrate with other analyses, e.g., methylation, copy number,
Correlation between genomic copy number and mRNA expression
identified 38 mis-labeled samples in the TCGA ovarian cancer
Affymetrix microarray dataset.
The lab is based on a modified version of the
RNA-seq work flow developed by Michael Love, Simon Anders, Wolfgang