Single molecule RNA sequencing uncovers trans-splicing and improves annotations in Anopheles stephensi

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Single molecule real-time (SMRT) sequencing provides full-length cDNA sequences, crucial for accurate genome annotation and RNA isoform discovery.
• The Asian malaria mosquito, Anopheles stephensi, is a significant vector of malaria, making its genome a key target for research.

Purpose of the Study:

• To utilize PacBio isoform sequencing to generate a comprehensive full-length cDNA library for Anopheles stephensi.
• To improve the gene annotation of Anopheles stephensi by leveraging high-accuracy, error-corrected sequencing data.
• To identify and characterize novel RNA splicing events, including trans-splicing, within the Anopheles stephensi genome.

Main Methods:

• Isoform sequencing (Iso-Seq) using Pacific Biosciences (PacBio) platform to generate full-length cDNA reads.
• Error correction of PacBio reads utilizing Illumina RNA sequencing (RNA-seq) data.
• Comparative genomics analysis to assess conservation of identified splicing events across mosquito species.

Main Results:

• Over 600,000 full-length cDNA sequences were identified from Anopheles stephensi.
• Error correction with Illumina RNA-seq yielded more comprehensive data compared to the default SMRT pipeline.
• Gene annotation was significantly enhanced, with 4867 gene models updated and 1785 new alternatively spliced isoforms discovered.
• Six trans-splicing events were identified in An. stephensi and found to be conserved in Anopheles gambiae and Aedes aegypti.

Conclusions:

• PacBio Iso-Seq, coupled with robust error correction, is a powerful tool for advancing genome annotation in complex organisms like Anopheles stephensi.
• The discovery of conserved trans-splicing events suggests a potential evolutionary mechanism for gene rescue or adaptation in mosquitoes.
• This study provides a significantly improved genomic resource for Anopheles stephensi, facilitating future research on malaria vector control.