Supplementary Methods RNA preparation and sequencing Rhinella marina specimens were collected from four locations across the invasive range in Australia in April/May 2013: two from the range edge (West) in the Kimberley region of Western Australia (El Questro, 16.007872S 128.020494E; Purnululu National Park, 17.433382S 128.301818E) and two from the range core (East) in Queensland (Innisfail, 17.496250S 146.046506E; Rossville, 15.705427S 145.222855E). Muscle tissue (triceps femoris) was harvested from five adult female individuals per location (Table S1) and immediately stored in RNAlater (Qiagen, USA) buffer to preserve the RNA integrity. Tissues were later transferred to 20 °C freezer for long-term storage. Approximately 30 – 50 mg of liquid nitrogen flash-frozen muscle tissue was carefully ground with a pestle and mortar. Total RNA was extracted from each sample using the PerfectPure RNA Fibrous Tissue Kit (5 PRIME, Hamburg, Germany) following the manufactures protocol. In addition, on column RNase-free DNase treatment was performed to ensure the removal of genomic DNA from the sample. Quality and concentration of total RNA for each sample were measured using an Agilent 2100 Bioanalyser (Agilent Technologies, USA) to ensure that >1 ug of total RNA was extracted (range 1 – 2.6 ug) and samples had high RNA Integrity Numbers (RIN values: range, 5.3 – 8.5; mean = 7.3). To each sample, we added 4 ul of a 1:100 dilution of either mix 1 or mix 2 of the External RNA Controls Consortium (ERCC) spikein control RNA (Life Technologies, USA) (Table S1). The addition of the ERCC ‘spike-ins’ serve as internal experimental controls and allow us to quantify the diagnostic performance of digital gene expression. Total RNA samples were prepared for sequencing using the Illumina TruSeq RNA (Illumina Inc. USA) protocol with the size selection step selecting for ~300 bp fragments and sequenced across 4 flow cell lanes of the Illumina HiSeq-2000 platform generating 101 bp paired-end reads; conducted commercially at Macrogen, South Korea. Samples from the four populations were 1 distributed across all flow cell lane to minimize technical bias during sequencing. Raw sequence reads in FASTQ format were deposited at the NCBI short read archive (SRA) under the BioProject number PRJNA277985. Raw read quality control, in silico normailsation and de novo transcriptome assembly Raw reads from one individual per population (RM0021M, RM0094M, RM0108M and RM0169M) were pooled to produce a representative read set across all populations and regions for de novo assembly. Pre-filtering raw reads for quality is vital to obtain an accurate, high quality assembly. First, raw reads containing adaptor sequences were trimmed with cutadapt v1.3 (Martin 2011). Second, reads were filtered based on quality scores (Phred), with reads discarded if 95% of bases across the read did not have a minimum Phred score of 30; performed using FASTX-Toolkit v0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit/). Next, we computed the GC content distribution for all reads in the dataset. Random hexamer priming is known to introduce a GC content bias in the first 13 bases of Illumina RNA-seq reads (Hansen et al. 2010), so to remove this bias we trimmed the initial 13 bases from the reads. Certain specific sequence motifs can produce false positive base call errors in Illumina data (Minoche et al. 2011; Allhoff et al. 2013) and assemblies using more than 60 million reads have been shown to accumulate errors in highly expressed genes (Francis et al. 2013). We adopted the strategy described in to remove systematic read errors and reduce read redundancy. Briefly, read errors were removed with the reptile error correction pipeline (Yang et al. 2010) using the following parameters after an initial optimisation run: kmerLen = 14, T_expGoodCnt = 2, T_card = 1, MaxBadQPerKmer = 6, Qlb = 67. Reads were then in silico normalised using, normalize-by-median.py with –C 20, -k 20 and –x 4e9 set as parameters, obtained from the khmer package (git://github.com/ged-lab/khmer.git)(Brown et al. 2012) which also requires screed (git://github.com/ged-lab/screed.git). The resulting errorcorrected and normalized reads were used to create a de novo assembly using the Trinity pipeline, r20140413p1 (Grabherr et al. 2011), with the default settings. 2 The non-normalised pooled set of reads were then mapped back to the de novo assembled transcripts using bowtie2 v2.1.0 (Langmead & Salzberg 2012) and abundance estimated using RSEM v1.2.14 (Li & Dewey 2011). We filtered out any transcripts with less that 1% of the pergene expression level using a script bundled with Trinity. These transcripts with low support are likely to be erroneous transcripts and assembly artifacts. Coding sequences (CDS) were predicted from the filtered transcripts using TransDecoder (http://transdecoder.sourceforge.net) to identify the longest CDS for a transcript. Functional Annotation Functional annotation was conducted using the Trinotate pipeline (http://trinotate.sourceforge.net). First, BLASTx and BLASTp searches of the filtered transcripts against the UniProtKB/Swiss-Prot database were conducted with blast v 2.2.26+ (Camacho et al. 2009), with an evalue cutoff of 1x 10-3. The presence of signal peptides and transmembrane domains were predicted with SignalP v4.1 (Petersen et al. 2011) and TMHMM v2.0c (Krogh et al. 2001), respectively. The resulting outputs were loaded into an SQLite database, eggNOG (Powell et al. 2012) and Gene Ontology (GO) (Ashburner et al. 2000) terms were annotated to BLASTp matches and a master annotation file was extracted. Reciprocal BLASTn searches of our assembled transcripts and the transcripts assembled by (Nourisson et al. 2014; Arthofer et al. 2015) were conducted to identify orthologous sequences between R. marina muscle and liver transcriptomes. Read library mapping, ERCC diagnostic performance and digital gene expression analysis The filtered transcripts set containing full and partial ORFs were used in the reference transcriptome to avoid skewing the expression quantification estimates with non-coding, fragmented and erroneous data along with transcript sequences for the 92 ERCC RNA spike-ins. Quality filtered reads from each individual sequencing library (20 R. marina samples in total) 3 were mapped to the reference using Bowtie and fragment abundance estimations, as read counts, were produced with RSEM. RSEM quantified expression counts were filtered for lowly expressed genes, with these removed when they had < 1 count per million reads (CPM) in n=10 or fewer libraries (where n is the smallest number of replicates per treatment). These genes have too low an expression to be detected as significantly differentially expressed between the two regions (East and West) and their inclusion reduces the power to detect other differentially expressed genes (Anders et al. 2013). To evaluate the technical performance of the differential expression experiment, expression values of the ERCC RNA spike-in ratios (mix 1 vs mix 2) contained within each individuals sequenced library were analysed using the erccdashboard R package (Munro et al. 2014). This program cannot compare both ERCC mix types within both regions concurrently. We therefore chose to examine experimental performance between the regions using mix1 samples from the West and mix 2 samples from the East as this aligned with our predicted gene expression differences and designed ERCC ratio fold-change differences. Diagnostic performance was evaluated with Receiver Operator Characteristic (ROC) curves and the Area Under the Curve (AUC) statistic, lower limit of differential expression detection estimates (LODR) and expression ratio variability and bias. These measures are based on the intrinsic ERCC transcript abundances that were added to the samples before sequencing and these external controls are essential to understand the validity of digital gene expression experiments. An AUC statistic of 1 represents prefect ability, while an AUC of 0.5 represent no reliability when calling differentially expressed genes. CPM filtered read counts for the ERCC sequences and reference genes were used as input for erccdashboard, with the number of mapped reads per sample used as normilisation factors and the following parameters: erccmix="RatioPair", erccdilution=1/100, spikeVol=2, totalRNAmass=1. These parameters result from the amount, type and dilution of the ERCC ratio mixes added to each 4 sample (Table S1). The diagnostic performance metrics were used to set our acceptable false discovery rates and p-values for calling endogenous differentially expressed genes. We used the BioConductor tool edgeR (Chen et al. 2014) in R (R Development Core Team 2011) to identify endogenous differentially expressed genes between the two range-edge (West) populations and the two core (East) populations. We adopted the classic approach for the comparison of two groups, East and West regions. CPM filtered counts were then TMMnormalized (Trimmed mean on M-values) (Robinson & Oshlack 2010), to account for library size and expression bias between replicates. The quantile-adjusted conditional maximum likelihood method was used to estimate dispersions and the exact test was used to call differentially expressed genes corrected for false discovery rate p < 0.05. We utilized scripts packaged with Trinity for further filtering of differentially expressed genes identified with edgeR, including filtering differentially expressed genes based on ERCC derived threshold p-values (p < 0.001) and minimum fold change (FC > 2; see Supplementary Results for ERCC metrics upon which these values were based), and for building MA plots. This provides a conservative cut off for evaluating significant differential gene expression between the range edge and core. The values chosen are based upon the intrinsic gene expression differences between the regions and are supported by the addition of true and false positives for the expression ranges provided by the ERCC controls (see Munro et al. 2014). Gene ontology enrichment analysis We used enrichment analysis to identify whether functional categories associated with an a priori suite of genes (those from the reference set used for assembly) contain more differentially expressed genes for each region than expected by chance. To account for the transcript length heterogeneity usually found in RNA-Seq data, which can bias enrichment analysis, we adopted the goseq method (Young et al. 2010), which is available as a BIoconductor R package. We extracted 5 the Gene Ontology (GO) categories (Biological Process, Molecular Function and Cellular Component) for genes from the de novo assembly Annotation Metatable (Table S4). Each set of GO categories for differentially expressed genes from the West and East regions comparison were assessed for enrichment compared to the reference set of GO categories when controlling for transcript length. Enrichment was determined using a Benjamini-Hochberg corrected p-value < 0.05. Significantly enriched GO categories were plotted using REVIGO (Supek et al. 2011) with the SimRel semantic clustering of similar GO functions when using the whole UniProt database to source annotations. Supplementary Results De novo assembly A total of 359,563,284 Illumina HiSeq reads were generated for the 4 samples used for de novo assembly. After trimming adaptors, the initial 13 bp of each read, quality filtering and digital normalization, 18,713,526 reads remained. These were assembled with Trinity into 82,411 transcripts representing 69,136 trinity components with an N50 of 1,604 bases. Abundance estimates for each transcript were generated by mapping the non-digitally normalized reads back to the assembly; transcripts with low read support were filtered out. This filtering removed 24,831 transcripts leaving 57,580 transcripts with an improved N50 of 1,871 bases (Table S2). When compared to a previous R. marina transcriptome assembly from liver tissue (Nourisson et al. 2014; Arthofer et al. 2015) our assembly has a similar N50 value but higher median and average contig lengths. Functional Annotation We predicted 19,751 peptide sequences from the filtered assembly of which 11,693 were predicted to have a complete CDS. In total we identified 21,533 transcripts with a significant BLASTx match (compares all 6 open reading frames of the nucleotide query sequence against the protein 6 database) and 16,754 transcripts with BLASTp matches (compares predicted protein sequences to the protein database) to proteins in the Swiss-Prot database (Table S3, Table S4). Transcripts without significant matches to the database could be from untranslated regions, non-coding RNAs, lack a protein domain, derive a protein that has no functional annotation or is not contained within the database. Signal peptides were predicted in approximately 2 % of transcripts while we identified transmembrane helices in 5.2 % of transcripts. We mapped Gene Ontology (GO) terms to 19,500 transcripts covering biological, cellular and molecular functions. We conducted a reciprocal BLASTn search of our assembled transcripts to those previously assembled for R. marina liver tissue and identified 28,790 orthologous transcripts (Table S5). This constitutes one of our muscle transcripts having a best match to a liver transcript where that liver transcript conversely has a best match to the same muscle transcript. As such, the annotations provided here can also serve as putative annotations for the orthologous liver transcripts, which currently have no functional information. Sequencing and read mapping for the range edge versus core regions Sequencing of cDNA libraries yielded between 68.8 and 97.6 million reads per sample (Table S1). After adapter trimming and quality filtering of reads we obtained between 38.3 and 54.5 million reads per sample that still contained both paired reads. All reads that passed this filtering step had average Phred scores > Q30, which corresponds to a base call accuracy of > 99.9 %. Between 90.01 % and 93.59 % of reads mapped to the combined 57,580 assembled and 92 ERCC transcripts. Expression was quantified as counts per million mapped reads (CPM) and transcripts from the reference set were filtered out when they had a CPM < 1 in n=10 or more samples across both regions. This resulted in expression estimates used for differential expression analysis for a total of 19,569 assembled transcripts. 7 Assessment of technical and diagnostic performance We assessed the diagnostic capabilities of the experiment by comparing the expression estimates of the assembled and ERCC transcripts between both the regions (see Methods and Munro et al. 2014 for more details). We are reliably able to detect gene expression differences across a dynamic range of just under 215 (Fig. S1a). This is less than the range of 220 for which the ERCC ratios are designed to detect, meaning we have insufficient evidence to quantify ERCC expression at low abundances. Comparison between the ERCC ratio signal vs average signal indicates a reasonably large variation in the ERCC ratio measurements as a function of dynamic range (Log(rm) 0.86 ± 0.22 weighted SE, Fig. S1c). This indicates a difference in the mRNA fractions between the samples containing mix 1 and mix 2, which could arise through technical variability between sample cDNA library construction or from endogenous differences in the fraction of genes expressed between the samples. The ROC analysis showed our data and analysis had high power to detect real endogenous differential gene expression as indicated by AUC statistics ranging between 0.91 and 1 for the 3 ratios showing differential expression (Fig. S1b). As expected, our diagnostic power increases with an increased fold change. The LODR analysis indicates that we can reliably detect expression differences as true positives at a threshold p-value < 0.05 for 2- and 4-fold changes (Fig. S1d). Based on these metrics we set a minimum fold change threshold of 2 and a p-value threshold of 0.001 for filtering endogenous differential expression. This criterion was adopted to account for any potential bias between the mRNA fractions of samples, reduce the occurrence of potential false positives and limit the analysis to those genes most significantly differentially expressed between the regions. Differential gene expression between the range edge and range core regions A pairwise comparison of CPM-filtered gene expression estimates for toads from the range edge (West) versus the range core (East) regions revealed a total of 3,449 significantly differentially expressed genes at an FDR of 0.05. When filtered based on our ROC/AUC and LODR metrics (p 8 = 0.001 and > 2-fold change), we retained 621 significant differentially expressed genes. Of these 479 were up-regulated in the Western region and the remaining 142 were down-regulated in the West compared to the Eastern range core (Fig. S2). 9 Literature cited Allhoff M, Schönhuth A, Martin M et al. (2013) Discovering motifs that induce sequencing errors. BMC bioinformatics, 14 Suppl 5, S1. Anders S, McCarthy DJ, Chen Y et al. (2013) Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nature Protocols, 8, 1765–1786. Arthofer W, Banbury BL, Carneiro M et al. (2015) Genomic Resources Notes Accepted 1 August 2014-30 September 2014. Molecular ecology resources, 15, 228–229. Ashburner M, Ball CA, Blake JA et al. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nature genetics, 25, 25–9. Brown CT, Howe A, Zhang Q, Pyrkosz AB, Brom TH (2012) A Reference-Free Algorithm for Computational Normalization of Shotgun Sequencing Data. , 1–18. Camacho C, Coulouris G, Avagyan V et al. (2009) BLAST+: architecture and applications. BMC bioinformatics, 10, 421. Chen Y, Mccarthy D, Robinson M, Smyth GK (2014) edgeR : differential expression analysis of digital gene expression data User ’ s Guide. Francis WR, Christianson LM, Kiko R et al. (2013) A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly. BMC genomics, 14, 167. Grabherr MG, Haas BJ, Yassour M et al. (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature biotechnology, 29, 644–52. Hansen KD, Brenner SE, Dudoit S (2010) Biases in Illumina transcriptome sequencing caused by random hexamer priming. Nucleic acids research, 38, e131. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of molecular biology, 305, 567–80. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nature methods, 9, 357–9. Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC bioinformatics, 12, 323. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal, 17, 10. Minoche AE, Dohm JC, Himmelbauer H (2011) Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and genome analyzer systems. Genome biology, 12, R112. Munro SA, Lund SP, Pine PS et al. (2014) Assessing technical performance in differential gene expression experiments with external spike-in RNA control ratio mixtures. Nature communications, 5, 5125. Nourisson C, Carneiro M, Vallinoto M, Sequeira F (2014) Data from: “De novo transcriptome assembly and polymorphism detection in ecological important widely distributed Neotropical toads from the Rhinella marina species complex (Anura: Bufonidade)” in Genomic Resources Notes Accepted 1 August 2014-30 September. Molecular Ecology Resources. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature methods, 8, 785–6. Powell S, Szklarczyk D, Trachana K et al. (2012) eggNOG v3.0: orthologous groups covering 1133 organisms at 41 different taxonomic ranges. Nucleic acids research, 40, D284–9. R Development Core Team R (2011) R: A Language and Environment for Statistical Computing (RDC Team, Ed,). R Foundation for Statistical Computing, 1, 409. Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome biology, 11, R25. Supek F, Bošnjak M, Škunca N, Šmuc T (2011) REVIGO summarizes and visualizes long lists of gene ontology terms. PloS one, 6, e21800. Yang X, Dorman KS, Aluru S (2010) Reptile: representative tiling for short read error correction. Bioinformatics (Oxford, England), 26, 2526–33. Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome biology, 11, R14. 10 Fig S1 (next page). ERCC technical and diagnostic plots produced by the erccdashboard. Each sample type contained n=10 biological replicates. A) Signal-abundance plot, points are coloured by ratio sub-pool, shape represents the sample type, and error bars denote the standard deviations of the replicates. B) ROC curves and AUC statistics for each group of true-positive ERCC controls (detected = the number of controls used and spiked = the total included in the ERCC control mixture. C) MA plot of ERCC ratio measurement variability and bias. Coloured data points represent the mean ratio measurement per ERCC transcript, error bars the standard deviation of the replicates ratios, and filled circles are ERCC ratios above the LODR estimates. Grey points denote endogenous transcript ratio measurements. Nominal ERCC ratios for each sub-pool are annotated with coloured solid lines, dashed lines represent the adjusted ratios based on the estimate of mRNA fraction differences between the samples, rm. D) LODR estimates are indicated by coloured arrows for each fold change that crosses the threshold p-value, the black dashed line denotes the threshold p-value derived for the chosen FDR. LODR results and bootstrap confidence interval are provided in the table below the plot. 11 A B Sample 1.00 mix1 mix2 10 Ratio 4:1 1:1 0.75 1:2 Ratio TPR Log2 Normalized ERCC Counts 1:1.5 5 0 4:1 0.50 1:1.5 1:2 −5 0.25 −10 Ratio AUC Detected Spiked 4:1 1.000 15 23 1:1.5 0.907 14 23 1:2 0.964 15 23 0.00 0 5 10 15 20 0.00 Log2 ERCC SpikeAmount(attomol nt/mg total RNA) 0.25 0.50 0.75 1.00 FPR C4 D Weighted Mean (+/−) Weighted Standard Error log(rm) 0.8609 1e+00 0.2236 3 1e−01 DE Test P−values Log2 Ratio of Normalized Counts 2 1 0 1e−02 1e−03 ● ● −1 ● ● ● ● ● ● ● ● ● 1e−04 ● ● ● ● ● ● ● ● ● −2 ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ●● ●● ● ● ●● ● ●●● ● ● ● ● ● ●● ● ●● ● ● ● ● Ratio ● ● ● ● 4:1 ● ● ● ● ● ● ● ● ● ● ● ● 1:1 ● ● 1:1.5 ● 1:2 ● Ratio 10 −3 1000 Average Counts 4:1 1:1 1:1.5 1:2 −4 ● ● ●● ● ● −10 −5 0 5 Log2 Average of Normalized Counts 10 Ratio LODR Estimate 90% CI Lower Bound 90% CI Upper Bound 4:1 7.1 <1.1 1:1.5 Inf NA 8.1 NA 1:2 2100 79 4100 12 Fig. S2 MA plot of differentially expressed genes identified between the range-core and range-edge. For each gene the log2 fold-change (y-axis) is plotted against the average log2 expression (x-axis) in counts per million mapped reads. Each dot represents a transcript and significant differential expression is indicated as red dots with at most 0.05% FDR. 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● ● ● ● ● ● ● ● ● logFC ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● 0 5 10 15 Average logCPM 13