Freddie: Annotation-independent Detection and Discovery of Transcriptomic Alternative Splicing Isoforms

Abstract Alternative splicing (AS) is an important mechanism in the development of many cancers, as novel or aberrant AS patterns play an important role as an independent onco-driver. In addition, cancer-specific AS is potentially an effective target of personalized cancer therapeutics. However, detecting AS events remains a challenging task, especially if these AS events are not pre-annotated. This is exacerbated by the fact that existing transcriptome annotation databases are far from being comprehensive, especially with regard to cancer-specific AS. Additionally, traditional sequencing technologies are severely limited by the short length of the generated reads, that rarely spans more than a single splice junction site. Given these challenges, transcriptomic long-read (LR) sequencing presents a promising potential for the detection and discovery of AS. We present Freddie, a computational annotation-independent isoform discovery and detection tool. Freddie takes as input transcriptomic LR sequencing of a sample and computes a set of isoforms for the given sample. Freddie takes as input the genomic alignment of the transcriptomic LRs generated by a splice aligner. It then partitions the reads to sets that can be processed independently and in parallel. For each partition, Freddie segments the genomic alignment of the reads into canonical exon segments. The goal of this segmentation is to be able to represent any potential isoform as a subset of these canonical exons. This segmentation is formulated as an optimization problem and is solved with a Dynamic Programming algorithm. Then, Freddie reconstructs the isoforms by jointly clustering and error-correcting the reads using the canonical segmentation as a succinct representation. The clustering and error-correcting step is formulated as an optimization problem – the Minimum Error Clustering into Isoforms (MErCi) problem – and is solved using Integer Linear Programming (ILP). We compare the performance of Freddie on simulated datasets with other isoform detection tools with varying dependence on annotation databases. We show that Freddie outperforms the other tools in its recall, including those given the complete ground truth annotation. In terms of false positive rate, Freddie performs comparably to the other tools. We also run Freddie on a transcriptomic LR dataset generated in-house from a prostate cancer cell line. Freddie detects a potentially novel Androgen Receptor isoform that includes novel intron retention. We cross-validate this novel intron retention using orthogonal publicly available short-read RNA-seq datasets. Availability Freddie is open source and available at https://bitbucket.org/baraaorabi/freddie

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