A Comprehensive Hybrid Transcriptome Assembly Reveals Coding and Non-coding Landscapes in Wasp-infested Drosophila Larvae

Drosophila larval hemocytes mount a potent immune response against parasitic wasps and a large, flat cells, the lamellocyte, is responsible for the response. Previous short-read based (single-cell) RNA sequencing has delineated protein-coding genes involved in lamellocyte differentiation, but incomplete transcript boundaries and uncharacterized long non-coding RNAs (lncRNAs) limit our understanding of these immune-regulatory networks. Here, we performed out a hybrid sequencing strategy integrating Oxford Nanopore long-read and Illumina short-read sequencing to provide a more comprehensive transcriptome annotation - enabling the discovery of novel coding and non-coding transcripts and elucidating alternative isoform usage during wasp infeatation. In particular, 349 unannotated long non-coding RNAs were discovered, some of which are highly induced at the late stages of wasp infestation. Full-length transcripts with novel 5' and 3' boundaries helped to reveal cell type-specific lncRNA markers in lamellocytes and crystal cells by single-cell analyses, which recapitulated hemocyte differentiation trajectories. Notably, RNAi-based depletion of two highly induced lncRNAs impaired lamellocyte formation under wasp infestation, highlighting their functional relevance. Collectively, our findings provide detailed insights into the Drosophila larval immune transcriptomes through the long-read sequencing and highlight the regulatory roles of non-coding RNAs in innate immunity.