Genome concatenation enables accurate dual RNA-seq mapping for lignocellulolytic fungi in coculture.

Dual RNA-seq enables simultaneous transcriptome profiling of interacting organisms, yet accurate read assignment remains challenging, particularly in fungal cocultures where phylogenetic proximity and high percentages of homologous sequences can be shared. Here, we evaluate two mapping strategies for dual RNA-seq analysis of Trichoderma reesei RUT-C30 and Pleurotus citrinopileatus cocultivated on sugarcane bagasse, based on a sequential alignment against individual genomes and a concatenated reference genome approach. Illumina NovaSeq sequencing generated 175 million high-quality reads, with mapping rates exceeding 96% across all strategies. No significant differences were observed between mapping strategies or genome order in the sequential approach (p = 0.976 and p = 1.0, respectively). Cross-mapping and multimapping rates were uniformly low (< 2% and < 1.7%, respectively), consistent with the substantial evolutionary divergence between the two fungi, and ensuring that read assignment to each genome was not significantly affected. The concatenated strategy reduced computational time by ~ 93% and yielded slightly more balanced genome-specific read assignments and lower multimapping frequencies. Together, these results demonstrate that concatenated genome mapping offers an efficient and accurate framework for resolving mixed fungal transcriptomes, appropriate for investigation of coculture systems and gene expression modulation during the degradation of lignocellulosic biomass.