Nanopore long-read RNA-seq and absolute quantification delineate transcription dynamics in early embryo development of an insect pest

Anthony Bayega,Yu Chang Wang,Spyros Oikonomopoulos,Kostas D Mathiopoulos,Jiannis Ragoussis,Maria-Eleni Gregoriou,Konstantina T Tsoumani,Antonis Giakountis

doi:10.1038/s41598-021-86753-7

Anthony Bayega, Yu Chang Wang + Show 6 more

Open Access

https://doi.org/10.1038/s41598-021-86753-7

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Abstract

The olive fruit fly, Bactrocera oleae, is the most important pest for the olive fruit but lacks adequate transcriptomic characterization that could aid in molecular control approaches. We apply nanopore long-read RNA-seq with internal RNA standards allowing absolute transcript quantification to analyze transcription dynamics during early embryo development for the first time in this organism. Sequencing on the MinION platform generated over 31 million reads. Over 50% of the expressed genes had at least one read covering its entire length validating our full-length approach. We generated a de novo transcriptome assembly and identified 1768 new genes and a total of 79,810 isoforms; a fourfold increase in transcriptome diversity compared to the current NCBI predicted transcriptome. Absolute transcript quantification per embryo allowed an insight into the dramatic re-organization of maternal transcripts. We further identified Zelda as a possible regulator of early zygotic genome activation in B. oleae and provide further insights into the maternal-to-zygotic transition. These data show the utility of long-read RNA in improving characterization of non-model organisms that lack a fully annotated genome, provide potential targets for sterile insect technic approaches, and provide the first insight into the transcriptome landscape of the developing olive fruit fly embryo.

Highlights

The olive fruit fly, Bactrocera oleae, is the most important pest for the olive fruit but lacks adequate transcriptomic characterization that could aid in molecular control approaches
We and others have shown that the Oxford Nanopore Technologies (ONT) MinION sequencing platform provides full-length transcript resolution[2,3] and enables identification of hitherto unknown genes and isoforms[4,5,6,7] and provides gene expression quantification that is comparable to current s tandards[4,5,6,7]
We use long-read RNA sequencing (RNA-seq) and absolute quantification to elucidate the transcription dynamics in the developing embryos of the olive fruit fly collected at hourly intervals for the first 6 h after egg laying (AEL)

Summary

Introduction

The olive fruit fly, Bactrocera oleae, is the most important pest for the olive fruit but lacks adequate transcriptomic characterization that could aid in molecular control approaches. We further identified Zelda as a possible regulator of early zygotic genome activation in B. oleae and provide further insights into the maternal-to-zygotic transition These data show the utility of long-read RNA in improving characterization of non-model organisms that lack a fully annotated genome, provide potential targets for sterile insect technic approaches, and provide the first insight into the transcriptome landscape of the developing olive fruit fly embryo. In Drosophila, this process starts ~ 1 h after fertilization (AF) and involves the clearance of up at least 35% of transcripts that are maternally deposited in the developing e gg[11] followed by activation of the zygotic genome (reviewed in Tadros and Lipshitz[11], and Langley et al.[12]) These changes are highly dynamic and tightly regulated, capturing them could inform environmentally friendly measures aimed at controlling pests. Many early embryonic genes and their promoter/enhancer regions have been under intensive studies since they are used in pest control approaches involving transgenic embryonic lethality[13,14]

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