RATIONALEThe growing recognition of the importance of splicing, together with rapidly accumulating RNA-sequencing data, demand robust high-throughput approaches, which efficiently analyze experimentally derived whole-transcriptome splice profiles.RESULTSWe have developed a computational approach, called SNPlice, for identifying cis-acting, splice-modulating variants from RNA-seq datasets. SNPlice mines RNA-seq datasets to find reads that span single-nucleotide variant (SNV) loci and nearby splice junctions, assessing the co-occurrence of variants and molecules that remain unspliced at nearby exon-intron boundaries. Hence, SNPlice highlights variants preferentially occurring on intron-containing molecules, possibly resulting from altered splicing. To illustrate co-occurrence of variant nucleotide and exon-intron boundary, allele-specific sequencing was used. SNPlice results are generally consistent with splice-prediction tools, but also indicate splice-modulating elements missed by other algorithms. SNPlice can be applied to identify variants that correlate with unexpected splicing events, and to measure the splice-modulating potential of canonical splice-site SNVs.AVAILABILITY AND IMPLEMENTATIONSNPlice is freely available for download from https://code.google.com/p/snplice/ as a self-contained binary package for 64-bit Linux computers and as python source-code.CONTACT[email protected] or [email protected]SUPPLEMENTARY INFORMATIONSupplementary data are available at Bioinformatics online.