LongPolyASE: an end-to-end framework for allele-specific gene and isoform analysis in polyploids using long-read...

Allele-specific expression analysis can reveal cis-regulatory differences (e.g., promoter variants, epigenetic changes) that cause imbalanced gene expression between haplotypes. Haplotype-resolved reference genomes and long-read RNA sequencing enable allele-specific expression analysis at gene and isoform-levels. However, existing tools are largely restricted to short-read RNA sequencing data and diploid organisms.

We developed LongPolyASE, an end-to-end computational framework for allele-specific gene and isoform expression analysis in diploid and polyploid organisms using long-read RNA sequencing, consisting of three components: Syntelogfinder, for identifying syntenic gene relationships and annotation inconsistencies; longrnaseq, for novel isoform discovery and haplotype-level quantification; and PolyASE, for statistical testing and visualization of allelic imbalance and isoform usage. We applied LongPolyASE to diploid rice, autotetraploid potato, allotetraploid rapeseed, and allooctoploid strawberry using Oxford Nanopore and PacBio long-read RNA-seq. The framework enabled identification of cis-regulatory variation, tissue-specific trans-regulatory effects, differential isoform usage, and haplotype-specific splicing differences. In addition, it facilitated the discovery of novel transcripts and genes with potential functional relevance in plant development.

LongPolyASE addresses a key methodological gap by enabling allele-specific expression analysis in polyploid organisms using long-read RNA sequencing. By combining haplotype-aware quantification with isoform-level resolution in a reproducible workflow, the framework provides a practical tool for plant researchers working with complex genomes. Its application to crop species highlights its potential to support the identification of regulatory variation and candidate targets for plant breeding.