Analysis of alternative splicing and polyadenylation of Ascosphaera apis genes based on third-generation long-read sequencing dataset

Abstract:

[Objective] Ascosphaera apis is a fungal pathogen that exclusively infects honeybee larvae, leading to chalkbrood, which is a chronic disease in beekeeping industry and results in heavy losses for apiculture. The objective of this work is to investigate alternative splicing (AS) and alternative polyadenylation (APA) of genes in A. apis mycelium (Aam) and spore (Aas) based on previously gained third-generation long-read sequencing dataset. [Methods] The type of AS events occurred in genes in Aam and Aaswas identified. The visualization of partial isoforms' structures was performed with IGV browser. APA sites of genes in Aam and Aas were identified using TAPIS pipeline. MEME software was used to investigate characteristics of sequences at 50 bp upstream of APA sites followed by identification of motifs. [Results] In total, 286 AS events were identified in Aam, including 162 retained intron (RI), 87 alternative 3ʹ splice-site (A3), 32 alternative 5ʹ splice-site (A5) and five skipping exon (SE), while 559 AS events were identified in Aas, including 305 RI, 155 A3, 85 A5, 13 SE and one mutually exclusive exon (MEE). Further analysis suggested that majority of annotated genes in current reference genome are incomplete, number and structure of partial annotated genes in mycelium differ from those in spore, and for part of isoforms, there are no corresponding annotated genes in reference genome. Additionally, a total of 2748 genes in Aam were observed to contain one and more APA sites, among them those containing one APA site was the largest group (726, 26.42%); while in Aas 2768 gene were found to contain one and more APA sites, and those containing more than five APA sites were the most abundant (1180, 42.63%). Besides, part of genes in A. apis mycelium and spore were detected to have various APA sites. Moreover, analysis of sequence characteristics indicated that upstream and downstream sequences of 3ʹ UTR of A. apis full-length transcripts have an obvious base bias, and U and A were respectively enriched in the upstream and downstream. Moreover, four motifs were identified at the upstream of APA sites of A. apis full-length transcripts, including UCUCCU, UCUUCU, CCCACC and CCCCCU. [Conclusion] In this study, AS and APA of genes in A. apis mycelium and spore were deeply analyzed, the results uncovered the complexity of A. apis transcriptome, offering valuable information for improvement of current genome and transcriptome annotations and a pivotal foundation for exploration of the function of AS and APA involved in regulation of A. apis gene expression.

Key words:long-read sequencing;Oxford Nanopore;Ascosphaera apis;alternative splicing;alternative polyadenylation