References Ran E, et al. (2013) Alternative cleavage and polyadenylation: extent, regulation and function. Nature Reviews Genetics 14, 496-506. Simpson JT, et al. (2009) ABySS: a parallel assembler for short read sequence data. Genome Research 19, 1117-1123. Robertson G. et al. (2010) De novo assembly and analysis of RNA-seq data. Nature Methods 7, 909-912. Trapnell C, et al. (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology 28, 511-515. Grabherr MG, et al. (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology 29, 644-652. Djebali S, et al. (2012) Landscape of transcription in human cells. Nature 489, 101-108. Abreviations 3’-end – Downstream end 5’-end – Upstream end ABySS – Assembly by short sequences APA – Alternative polyadenylation CDS – Coding DNA sequence CS – Cleavage site DNA – Deoxyribonucleic acid ENCODE – Encyclopedia of DNA elements EST – Expressed sequence tag FDR – False discovery rate PET – Paired-end tag Poly(A) – Polyadenylation PPV – Positive predictive value RNA – Ribonucleic acid RNA-Seq – Whole transcriptome shotgun sequencing ROC – Receiver operator characteristic SPAT – Searching for poly(A) tails TPR – True positive rate Trans-ABySS – Transcriptome ABySS UTR – Untranslated region WIG – Wiggle format 1. Overview Acknowledgements In cancers, alternative splicing and polyadenylation (APA) can affect transcript stability, transport and translation, and can change a transcript’s translated sequence. APA can be characterized with short-read sequencing using specialized library construction methods. For large-scale disease studies, however, it is desirable to characterize splicing and APA from a single library construction and sequencing run. In the work reported here, we describe SPAT, an analysis tool that uses de novo assembly of RNA-Seq data to Search for Poly(A) Tails. SPAT is designed to accept contigs from a range of de novo transcriptome assemblers. When used with Trans- ABySS, the overall pipeline reports fusions and rearrangements, indels, alternative splicing, and a range of types of APA. SPAT: Searching for Poly(A) Tails in RNA-Seq de novo Assemblies Raymond AGJ, Robertson AG, Chiu R, Mungall K, Nip KM, Kreitzman M, Jackman SD, Karsan A and Birol I 2. RNA-Seq Assembly and Poly(A) Tail Detection RNA-PET Clusters Using data supplied by ENCODE for cell line A459, we first thresholded peaks in RNA-PET coverage tracks, then clustered peaks that occurred within 100bp of each other. We used the resulting clusters as ‘true’ events for validations and ROC-like curves. ROC-Like Curve true positive | An RNA-PET cluster with at least one CS call within 100bp. false positive | A CS call that is not within 100bp of an RNA-PET cluster. true negative | Cannot be defined. false negative | An RNA-PET cluster with no CS call within 100bp. 3. Comparing Cleavage Site Calls RNA-Seq Tools Trans-ABySS/SPAT | Assemble with Trans-ABySS and process with SPAT. Trinity/SPAT | Assemble with Trinity and process with SPAT. Cufflinks | Assemble with Cufflinks and call CSs at transcript ends. ROC-like curves, which were insensitive to thresholds for RNA-PET peak detection and clustering, show that SPAT works with other assemblers, and performs better than a method that uses annotations to determine ends of expressed 3’ UTRs. SPAT has a PPV of 90% when we require an RNA-PET coverage of 3 and 2 bridging reads supporting a CS call. Validation Counts Venn diagrams were generated with the true positives from each tool set. Increasing RNA-PET stringency reduces the number of true positives by 6% for Trans-ABySS/SPAT and 27% for Cufflinks. We thank the ENCODE consortium for supplying the resources that made this analysis possible. We gratefully acknowledge funding from Genome BC, Genome Canada, and the BC Cancer Foundation. Trans-ABySS Assembly Assembly involves constructing