De novo assembly and characterization of the liver transcriptome of Mugil incilis (lisa) using next generation sequencing

Abstract

Mugil incilis (lisa) is an important commercial fish species in many countries, living along the coasts of the western Atlantic Ocean. It has been used as a model organism for environmental monitoring and ecotoxicological investigations. Nevertheless, available genomic and transcriptomic information for this organism is extremely deficient. The aim of this study was to characterize M. incilis hepatic transcriptome using Illumina paired-end sequencing. A total of 32,082,124 RNA-Seq read pairs were generated utilizing the HiSeq platform and subsequently cleaned and assembled into 93,912 contigs (N50 = 2,019 bp). The analysis of species distribution revealed that M. incilis contigs had the highest number of hits to Stegastes partitus (13.4%). Using a sequence similarity search against the public databases GO and KEGG, a total of 7,301 and 16,967 contigs were annotated, respectively. KEGG database showed genes related to environmental information, metabolism and organismal system pathways were highly annotated. Complete or partial coding DNA sequences for several candidate genes associated with stress responses/detoxification of xenobiotics, as well as housekeeping genes, were employed to design primers that were successfully tested and validated by RT-qPCR. This study presents the first transcriptome resources for Mugil incilis and provides basic information for the development of genomic tools, such as the identification of RNA markers, useful to analyze environmental impacts on this fish Caribbean species.

Figure 1

Function classifications of Gene Ontology (GO) terms of M. incilis transcriptome data. GO terms were annotated at level 2 of classification according to three main categories (Biological process, Cellular component and Molecular function). The x-axis indicates the subcategories, and the y-axis registers the percentages of genes (number of a particular gene divided by total gene number).

Figure 2

Top 20 pathways with the largest numbers of contigs based on KEGG annotation analysis.

Figure 3

Gel electrophoresis of the RT-PCR products of genes associated with stress responses/xenobiotic detoxification. The lanes correspond to blank (1), cDNA from M. incilis (2–9), P. magdalenae (11–12), H. malabaricus (13–14) and M. musculus (15). All the gels were run under the same experimental conditions and are presented using cropped images. The entire gel photos of all genes evaluated can be found in Figure S5.