Pipeline flow chart. Flow chart depicting the steps in the pipeline and two main use cases. (a) expression estimation using a pre-defined transcriptome reference; (b) construction of a custom transcriptome reference from the data followed by expression estimation. Haplotype-specific expression can be obtained using a pre-defined transcript reference if the parental transcriptome sequences are known and recombination has no effect (for example in the case of an F1 cross of two inbred strains). If the standard (for example Ensembl) reference is used, then isoform-level estimates are produced. If a custom reference is constructed solely to avoid allelic mapping biases, the phasing of genotypes can be omitted and isoform-level estimates are produced. If the genotypes are phased, haplo-isoform estimation is performed.