SMRT sequencing of full-length transcriptome of flea beetle Agasicles hygrophila (Selman and Vogt)

Dong Jia,Lingling Gao,Yuanxin Wang,Yanhong Liu,Yanqiong Guo,Ruiyan Ma,Jun Hu

doi:10.1038/s41598-018-20181-y

Abstract

This study was aimed at generating the full-length transcriptome of flea beetle Agasicles hygrophila (Selman and Vogt) using single-molecule real-time (SMRT) sequencing. Four developmental stages of A. hygrophila, including eggs, larvae, pupae, and adults were harvested for isolating total RNA. The mixed samples were used for SMRT sequencing to generate the full-length transcriptome. Based on the obtained transcriptome data, alternative splicing event, simple sequence repeat (SSR) analysis, coding sequence prediction, transcript functional annotation, and lncRNA prediction were performed. Total 9.45 Gb of clean reads were generated, including 335,045 reads of insert (ROI) and 158,085 full-length non-chimeric (FLNC) reads. Transcript clustering analysis of FLNC reads identified 40,004 consensus isoforms, including 31,015 high-quality ones. After removing redundant reads, 28,982 transcripts were obtained. Total 145 alternative splicing events were predicted. Additionally, 12,753 SSRs and 16,205 coding sequences were identified based on SSR analysis. Furthermore, 24,031 transcripts were annotated in eight functional databases, and 4,198 lncRNAs were predicted. This is the first study to perform SMRT sequencing of the full-length transcriptome of A. hygrophila. The obtained transcriptome may facilitate further exploration of the genetic data of A. hygrophila and uncover the interactions between this insect and the ecosystem.

Highlights

Alligator weed Alternanthera philoxeroides (Mart.) (Amaranthaceae) that originated from South America[1,2], was introduced into China in the 1930 s
The eggs laid by A. hygrophila within 24 h, first- to third-instar larvae starved for 24 h, pupae (2–6 days old), and newly emerged adults starved for 24 h were gathered and rinsed three times in precooled normal saline
The methodological strengths of single-molecule real-time (SMRT) sequencing have been comprehensively investigated in human[13], which is superior to methods of short read sequencing due to the advantage of obtaining full-length transcripts

Summary

Introduction

Alligator weed Alternanthera philoxeroides (Mart.) (Amaranthaceae) that originated from South America[1,2], was introduced into China in the 1930 s. Due to the good performances in host selection and ecological adaptation, A. hygrophila has become a desirable model insect for the investigation of the relationships between insects and plants, as well as insects and ecosystem. Short-read transcriptome sequencing has become a powerful tool for the description of gene expression levels[10,11]. Most of these technologies are incapable of assembling full-length transcripts because of the shortness of sequencing reads, which necessitates efforts for exploring other technologies. SMRT sequencing was performed to generate full-length transcriptome of A. hygrophila. This study may be a valuable resource for further investigation of A. hygrophila

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Conclusion