De novo transcriptome analysis by deep RNA sequencing

Abstract

For researchers using non-model animal species, we here introduce the method of de novo transcriptome sequencing using Illumina short read data at a low cost. Sequence assembly without a reference genome is called de novo assembly. The absence of genome information has been a large disadvantage for transcriptome sequencing of non-model animals, because it makes transcriptome assembly difficult. The present technical note describes how to prepare the RNA sample, perform a deep RNA sequencing and de novo assembly. For de novo assembly, five assembly programs are compared: ABySS, Velvet, OASES, Trinity and Rnnotator. We also show how to classify the obtained sequences associated with gene ontology (GO) terms at the protein level annotation using BLAST2GO program. We hope this technical note will help the researchers to start a transcriptome analysis and facilitate the future genetic analysis of non-model animal.