SUPPLEMENTARY INFORMATION
Todd, Black and Gemmell. The power and promise of RNA-seq in ecology and evolution.
Appendix S1
References reviewed
Details of references reviewed in order to evaluate trends in biological replicate usage by ecoevolutionary studies utilising RNA-seq data for differential expression analysis (2008-2014)
(Box 1). For each of 158 studies reporting statistical differential expression analysis from
RNA-seq data, the table below gives the focal organism, sequencing platform, and details of
the experimental design, including the number of treatment groups, whether or not multiple
samples were pooled for library preparation, and the number of biological replicates used (as
the number of replicate library preparations per treatment group).
3
Pooled/separate
library preparation
Pooled
Biological
replicates
2
Illumina
2
Pooled
1
454
Illumina
Illumina
2
4
4
Pooled
Separate
Separate
1
4, 5 or 6
4 to 6
Illumina
9
Separate
1
Illumina
54
Separate
2
Acyrthosiphon pisum
Illumina
2
Pooled
2
Microplitis demolitor
Illumina
4
Pooled
1
Saccharomyces spp.
Ceratitis capitata
Aphis glycines
Illumina
Illumina
Illumina
4
6
6
Separate
Pooled
Pooled
2
3
4
Polygonia c-album
Illumina
4
Pooled
2
Illumina
Illumina
454
2
12
2
Separate
Pooled
Pooled
10
1
1
SOLiD
2
Separate
1
Illumina
6
Separate
6 to 10
Illumina
8
Pooled
1 or 2
Illumina
3
Separate
2, 3 or 4
Paper
Year
Subject
Platform
Conditions
Arfi et al. 2013
2013
Pycnoporus coccineus
Sarotherodon
melanotheron heudelotii
Hyriopsis cumingii
Acropora hyacinthus
Symbiodinium spp.
Symbiodinium
microadriaticum
multiple
Illumina
Avarre et al. 2014
Bai et al. 2013
Barshis et al. 2013
Barshis et al. 2014
Baumgarten et al.
2013
Brawand et al. 2011
Burke and Moran
2011
Burke and Strand
2014
Busby et al. 2011
Calla et al. 2014
Cassone et al. 2014
Celorio-Mancera et
al. 2013
Chapman et al. 2013
Chen et al. 2010
Chen et al. 2011
2014
2013
2013
2014
2013
2011
2011
2014
2011
2014
2014
2013
2013
2010
Chu et al. 2014
2014
Senecio spp.
Locusta migratoria
Balanus amphitrite
Methanolobus
psychrophilus R15
Peromyscus maniculatus
Brugia malayi, Aedes
aegypti
Nucella lapillus
Coate et al. 2013
2013
Glycine dolichocarpa
Illumina
12
Pooled
3
2013
multiple
Amphimedon
queenslandica
Illumina
4
Pooled
1
SOLiD
4
Pooled
1
Trichoderma harzianum
Illumina
3
Separate
1
Separate
3
Chen et al. 2012
Cheviron et al. 2014
Choi et al. 2014
Colombo et al. 2013
Conaco et al. 2012
2011
2012
2014
2014
2012
Crivelente Horta et
al. 2014
Czesny et al. 2012
2014
2012
Alosa pseudoharengus
454
2
Daniels et al. 2014
2014
Junonia coenia
Illumina
8
Pooled
2
Darby et al. 2014
2014
Wolbachia
Illumina
2
Separate
3
de Groot et al. 2014
Denekamp et al.
2011
Dion-Cote et al.
2014
Dirks et al. 2014
2014
Deinococcus deserti
Illumina
2
Separate
1
Brachionus plicatilis
Illumina
13
Pooled
1
lake whitefish
Illumina
5
Pooled
4 or 5
Anguilla anguilla
Illumina
4
Separate
3 or 4
2011
2014
2014
Dong et al. 2014
2014
Bactrocera minax
Illumina
4
Dou et al. 2013
Separate
1
2013
Liposcelis bostrychophila
Illumina
3
Pooled
1
Dowen et al. 2012
2012
Arabidopsis thaliana
Illumina
4
Separate
2
Duncan et al. 2014
2014
multiple
Illumina
2
Pooled
1
Dunning et al. 2013
2013
Micrarchus nov. sp. 2
454
2
Separate
2
Dunning et al. 2014
2014
Micrarchus spp.
Illumina
6
Separate
3 or 6
Ekblom et al. 2012
2012
Philomachus pugnax
454
6
Separate
3 to 6
Ekblom et al. 2014
2014
Passer domesticus
Illumina
3
separate
1
Elran et al. 2014
Esponosa et al.
2014
Etebari et al. 2011
Eves-van den Akker
et al. 2014
Fan et al. 2013
2014
Nematostella vectensis
Illumina
5
Pooled
3 or 4
Perkinsus marinus
Illumina
2
Separate
3
Plutella xylostella
Illumina
2
Pooled
1
Nacobbus aberrans
Illumina
3
Pooled
3
2013
Salicornia europaea
Illumina
12
Pooled
1
Fang et al. 2013
Feldmeyer et al.
2014
Ferreira et al. 2013
2013
Caulobacter crescentus
Temnothorax
longispinosus
Polistes canadensis
SOLiD
5
Separate
3
Illumina
4
Pooled
1
Forconi et al. 2014
Franssen et al. 2014
2014
2011
2014
2014
2013
2014
2014
Fraser et al. 2014
Garcia-Vedrenne et
al. 2013
Gerdol et al. 2014
2014
Grumaz et al. 2013
2013
2014
illumina
4
Separate
2 to 6
Latimeria spp.
Zostera marina,
Nanozostera noltii
Poecilia latipinna
Illumina
3
separate
1
Illumina
8
Pooled
1
Illumina
4
Separate
2 or 3
Quahog Parasite Unknown
Illumina
2
Pooled
1
Mytilus galloprovincialis
Illumina
4
Pooled
1
2013
Candida spp.
Illumina
4
Separate
1 or 3
Gunter et al. 2013
2013
Astatoreochromis alluaudi
Illumina
2
Separate
5
Hanson et al. 2013
2013
Brachionus calyciflorus
Illumina
2
Pooled
2
Harr et al. 2010
2010
Mus spp.
Illumina
4
Separate
2
Henning et al. 2013
Hershkovitz et al.
2013
Hook et al. 2014a
2013
Amphilophus citrinellus
illumina
3
Separate
6
Metschnikowia fructicola
Illumina
4
Pooled
1
2014
Ceratoneis closterium
454
4
Pooled
1
Hook et al. 2014b
2014
Melita plumulosa
454
4
Pooled
1
Huth and Place 2013
Ibarra-Laclette et al.
2011
Ignatov et al. 2010
2013
Trematomus bernacchii
454
6
Pooled
1
Utricularia gibba
454
4
Pooled
1
2010
Mycobacterium avium
454
2
Pooled
1
Jeukens et al. 2010
2013
2011
2010
Coregonus clupeaformis
454
2
Pooled
1
Kanter et al. 2013
2013
Ambrosia artemisiifolia
454
2
Pooled
1
Kao et al. 2013
2013
Schmidtea mediterranea
SOLiD
12
Pooled
2
Kelley et al. 2012
2012
Poecilia mexicana
Illumina
2
Separate
3
Kersten et al. 2013
2013
Quercus rober L.
Illumina
4
Pooled
1
Kim et al. 2013
2013
Pseudomonas putida
Illumina
4
Separate
1
Kimura et al. 2011
Kobayashi et al.
2013
Kodama et al. 2014
2011
Dokdonia sp. MED134
454
3
Separate
1
Shorea beccariana
454
4
Separate
1
Paramecium bursaria
Illumina
2
Separate
3
Kumaran et al. 2014
2013
2014
2014
Bactrocera tryoni
Illumina
2
Separate
1
Lee et al. 2013
2013
Laodelphax striatellus
454
2
Pooled
1
Lei, Y. et al. 2014
2014
Plutella xylostella
Illumina
3
Separate
1
Lei, B. et al. 2014
2014
Nicotiana tabacum
Illumina
10
Pooled
1
Li et al. 2013
2013
Chrysopa pallens
Illumina
10
Pooled
1
Li et al. 2012
2012
Cnaphalocrocis medinalis
Illumina
5
Pooled
1
Liu et al. 2014
2014
Ostrinia furnacalis
Illumina
2
Pooled
1
2013
Vicia sativa
Illumina
8
Pooled
1
Fagopyrum spp.
454
2
Pooled
1
Oxyrrhis marina
Pyganodon grandis,
Uniomerus tetralasmus
Portunus trituberculatus
454
2
Pooled
1
Illumina
4
Pooled
3
Illumina
3
Pooled
1
Amphilophus citrinellus
Illumina
6
Separate
4, 5 or 6
Liu et al. 2013
Logacheva et al.
2011
Lowe et al. 2011
Luo et al. 2014
2011
2011
2014
Lv et al. 2013
Manousaki et al.
2013
Marra et al. 2014
2013
2014
multiple
Illumina
3
Separate
4
Mayfield et al. 2014
McDonnell et al.
2013
Meng et al. 2013
2014
Pocillopora damicornis
Illumina
4
Separate
3
Apis mellifera
Illumina
3
Pooled
2
2013
Sinocyclocheilus spp.
Illumina
4
Pooled
1
Meyer et al. 2011
Moyers and
Rieseberg 2013
Newton et al. 2013
2011
Acropora millepora
454
6
Pooled
1
Helianthus annuus
Illumina
2
Separate
6
2013
Lates calcarifer
Illumina
4
Pooled
1
Ng et al. 2014
2014
Illumina
5
Pooled
1
Illumina
2
Pooled
4
2013
2013
2013
2014
Gallus gallus
Nothobranchius furzeri, N.
kadleci
Salvelinus alpinus
Illumina
4
Separate
3
Novo et al. 2013
2013
multiple
Illumina
3
Separate
1
Nowack et al. 2011
2011
Paulinella chomatophora
Illumina
2
Separate
1
2012
multiple
454
4
Pooled
1
Palumbi et al. 2014
2014
Acropora hyacinthus
Illumina
2
Separate
6
Parikh et al. 2010
Pereira Lima
Teixeria et al. 2014
Perry et al. 2014
2010
Dictyostelium spp.
Illumina
2
Separate
2
Theobroma cacao
Illumina
2
Separate
5
Ng'oma et al. 2014
Norman et al. 2014
Ozawa et al. 2012
2014
2014
2014
Drosophila melanogaster
Illumina
6
Pooled
3
Pinosio et al. 2014
2014
Pinus halepensis
Illumina
2
Separate
1
Poelchau et al. 2011
2011
Aedes albopictus
454
2
Pooled
1
Poelchau et al. 2013
2013
Aedes albopictus
Illumina
6
Pooled
2 or 3
Poelchau et al. 2014
Prasopdee et al.
2014
Pravosudov et al.
2013
Reinhardt et al. 2014
Rendón-Anaya et al.
2012
Rinker et al. 2013
Sarropoulou et al.
2014
Schoville et al. 2012
2014
Aedes albopictus
Bithynia siamensis
goniomphalos
Illumina
4
Pooled
3
Illumina
2
Pooled
1
Poecile atricapillus
Illumina
2
Pooled
5
Teleopsis dalmanni
Centruroides noxius
Hoffmann
Anopheles spp.
Illumina
2
Pooled
2
454
2
Pooled
1
Illumina
2
Pooled
1
Sarda sarda
Illumina
5
Pooled
1
2014
2013
2014
2012
2013
2014
2012
Tigriopus californicus
Illumina
4
Pooled
1
Schunter et al. 2014
Schwarzenberger et
al. 2014
Seto and Tamura
2013
Shelomi et al. 2014
2014
Tripterygion delaisi
Illumina
3
Separate
5
Daphnia
Illumina
3
Pooled
3
Drosophila spp.
454
4
Pooled
1
2014
Peruphasma schultei
Illumina
2
Pooled
3
Shiao et al. 2012
2014
2013
2012
Mus musculus
Illumina
2
Pooled
1
Sikhakolli et al. 2012
2012
Fusarium spp.
Illumina
12
Separate
1
Sloan et al. 2014a
2014
Pachypsylla venusta
Illumina
2
Pooled
3
Small et al. 2013
2013
Smith, G. et al. 2014
Syngnathus spp.
454
4
Pooled
1
2014
Manduca sexta
Illumina
2
Pooled
4
Smith, G. et al. 2013
2013
Drosophila mojavensis
SOLiD
4
Pooled
4
Smith, S. et al. 2013
Somervou et al.
2014
Szoevenyi et al.
2011
Szoevenyi et al.
2013
Traeger et al. 2013
2013
Melanotaenia duboulayi
Illumina
2
Separate
6
Melitaea cinxia
Illumina
4
Separate
15
Funaria hygrometrica
454
2
Separate
1
Funaria hygrometrica
Illumina
6
Pooled
1
2013
multiple
Illumina
3
Pooled
2
2013
Ficedula albicollis
Illumina
16
Separate
5
Artemia franciscana
454
2
Pooled
1
Gluconacetobacter
diazotrophicus
Illumina
8
Pooled
1
Gryllus firmus
Illumina
3
Pooled
3
Pocillopora damicornis
Illumina
4
Pooled
1
2014
soybean
SOLiD
11
Pooled
1
Illumina
Uebbing et al. 2013
Valenzuela-Miranda
et al. 2014
Vargas et al. 2014
Vellichirammal et al.
2014
Vidal-Dupiol et al.
2013
Wang, L. et al. 2014
Wang, X. et al. 2014
2014
2011
2013
2014
2014
2014
2013
2014
Locusta migratoria
10
Pooled
1
Wang, Z. et al. 2014
2014
Lupinus albus
Illumina
3
Pooled
1
Wei et al. 2013
2013
Barbarea vulgaris
Illumina
7
Pooled
1
2013
Acropora millepora
Illumina
3
Separate
4
Scaptomyza flava
Illumina
4
Pooled
2
454
2
Separate
6
Illumina
7
Pooled
3
Weiss et al. 2013
Whiteman et al.
2012
Wolf et al. 2010
2012
Xiang et al. 2010
2010
Corvus corone
Heliocidaris
erythrogramma
Lateolabrax japonicus
Illumina
2
Pooled
1
Xie et al. 2014
2014
Bemisia tabaci
Illumina
8
Pooled
1
Xu et al. 2012
2012
Sogatella furcifera
Illumina
2
Pooled
1
Xu et al. 2013
2013
Leuciscus waleckii
Illumina
6
Pooled
1
Xu et al. 2014
2014
Oryza sativa
Illumina
5
Pooled
1
Xue et al. 2010
2010
Nilaparvata lugens
Illumina
6
Pooled
1
Wygoda et al. 2014
2010
2014
Yang et al. 2014
2014
Phalaenopsis
Illumina
2
Separate
1
Yeaman et al. 2014
2014
multiple
Illumina
14
Separate
3 to 8
Yek et al. 2013
2013
Acromyrmex echinatior
Illumina
5
Pooled
3
Yoshida et al. 2014
2014
Loligo bleekeri
Illumina
2
Pooled
1
Zhai et al. 2013
2013
Nilaparvata lugens
Illumina
4
Pooled
1
Zhang et al. 2011
2011
Xiphophorus maculatus
454
2
Pooled
1
Zhao et al. 2013
2013
Helicoverpa armigera
Illumina
2
Pooled
1
Zheng et al. 2012
2012
Bactrocera dorsalis
454
3
Pooled
1
Zheng et al. 2013
2013
Nannochloropsis spp.
Illumina
2
Separate
1
2014
Apostichopus japonicus
Illumina
4
Pooled
1
2014
Solanum lycopersicum
Illumina
2
Pooled
2
Zhou et al. 2014
Zouari et al. 2014
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Appendix S2
R code used for power analysis
R code and parameters used to perform power simulations on pilot RNAseq data from
bluehead wrasse (Box 3). Simulations were performed using the R/Bioconductor package
PROPER (Wu et al. 2015), with dispersion and baseline expression estimated from pilot data
via the DSS package (Wu et al. 2013). Pilot data consisted of two separate experiments
examining sex-biased gene expression in the gonad and forebrain of bluehead wrasse, each
experiment consisting of three biological replicates per sex (3 female, 3 male). Gonad and
forebrain experiments were analysed separately using identical simulation parameters, except
for the proportion of simulated differential expression. This was 25% for gonad, and 1% for
brain, chosen to reflect observations in real 3x3 comparisons for these tissues (Liu et al. in
review). Using the same value for this parameter did not alter the interpretation.
##Load counts table and define treatment groups.
wrasse<-read.table('rawcounts_table.tsv',header=T)
groups = c(rep("F",3), rep("M",3))
##Filter transcripts with fewer than 10 counts across all samples.
##Use DSS package to estimate dispersion and baseline expression.
library(DSS)
library(edgeR)
counts = as.matrix(wrasse)
counts<-counts[apply(counts,1,sum)>9,]
design = data.frame(gender=groups)
X = model.matrix(~gender, data=design)
rownames(X)<-colnames(counts)
seqData = newSeqCountSet(counts, as.data.frame(X))
seqData = estNormFactors(seqData)
seqData = estDispersion(seqData)
dispersion = dispersion(seqData)
fit.edgeR<-glmFit(counts, X, lib.size=normalizationFactor(seqData),
dispersion=dispersion(seqData))
lrt.edgeR<-glmLRT(fit.edgeR, coef=2)
##Simulate counts using PROPER package: 50,000 transcripts (‘ngenes’), 25% DE in gonad OR 1%
DE in brain (‘p.DE’).
##Simulate for 2, 3, 5, 7 and 10 replicates per condition (‘Nreps’), test DE with DESeq
(‘DEmethod’), over 100 iterations (‘nsims’).
library(PROPER)
library(DESeq)
sz = 50000
zz = sample(1:nrow(counts),sz)
sim.opts.wrasse = RNAseq.SimOptions.2grp(
ngenes = sz,
p.DE=0.01,
lOD = log(dispersion(seqData))[zz],
lBaselineExpr = apply(log(counts+0.5),1,mean)[zz],
lfc = lrt.edgeR$table$logFC[zz],
sim.seed = 11111)
simres = runSims(Nreps = c(2, 3, 5, 7, 10), sim.opts=sim.opts.wrasse,
DEmethod="DESeq", nsims=100)
##From simulated data, calculate power at FDR 0.05, for a 1.5 fold-change (equiv. 0.585 log2
fold-change) (‘delta’).
powers = comparePower(simres, alpha.type="fdr", alpha.nominal=0.05,
stratify.by="expr", delta=0.585)
summaryPower(powers)
##Plot result
plotPower(powers, main="Forebrain", lty=1,1:ncol(powers$power))