The study introduces an algorithm called Cufflinks for transcript assembly and abundance estimation from RNA-Seq data. Cufflinks is implemented in an open-source software program and was tested on a mouse myoblast cell line representing a differentiation time series. The algorithm detected 13,692 known transcripts and 3,724 previously unannotated transcripts, with 62% supported by independent expression data or homologous genes in other species. Analysis revealed complete switches in the dominant transcription start site (TSS) or splice-isoform in 330 genes and more subtle shifts in 1,304 genes, highlighting regulatory flexibility and complexity in muscle development. Cufflinks uses a statistical model to estimate transcript abundances, incorporating the distribution of fragment lengths to help assign fragments to isoforms. The software is applicable to a broad range of RNA-Seq studies and can be used to annotate genomes of newly sequenced organisms.The study introduces an algorithm called Cufflinks for transcript assembly and abundance estimation from RNA-Seq data. Cufflinks is implemented in an open-source software program and was tested on a mouse myoblast cell line representing a differentiation time series. The algorithm detected 13,692 known transcripts and 3,724 previously unannotated transcripts, with 62% supported by independent expression data or homologous genes in other species. Analysis revealed complete switches in the dominant transcription start site (TSS) or splice-isoform in 330 genes and more subtle shifts in 1,304 genes, highlighting regulatory flexibility and complexity in muscle development. Cufflinks uses a statistical model to estimate transcript abundances, incorporating the distribution of fragment lengths to help assign fragments to isoforms. The software is applicable to a broad range of RNA-Seq studies and can be used to annotate genomes of newly sequenced organisms.