Fig. 1

Workflow of Uncovering novel MHC alleles from RNA-Seq data (DinoMfRS). DinoMfRS enables the identification of previously unknown variant MHC alleles from RNA-Seq data through the creation of individual allele databases and a two-step approach that combines alignment of RNA-Seq reads to reference sequences and de novo assembly. The initial reference database (top left) consists of all known sheep MHC class I alleles. RNA-Seq reads (top right) are aligned to the class I reference sequences (using BWA-MEM). Known alleles carried by each individual show complete coverage, and the aligned RNA reads show no mismatches. From alignments with high coverage but mismatches to the reference allele (possibly novel alleles) RNA-Seq reads are extracted and de novo assembled using cap3 for further analysis. Consensus sequences from full-length cap3 assemblies are exported. An intermediate reference database containing the individual alleles will be created from these potentially novel alleles and the previously known alleles. A final BWA-MEM run is used to verify the novel alleles. The individual alleles of the examined sheep then result from the alignments showing complete coverage without mismatches