RNA-Seq Analysis and Gene Discovery of Andrias davidianus Using Illumina Short Read Sequencing

Abstract

The Chinese giant salamander, Andrias davidianus, is an important species in the course of evolution; however, there is insufficient genomic data in public databases for understanding its immunologic mechanisms. High-throughput transcriptome sequencing is necessary to generate an enormous number of transcript sequences from A. davidianus for gene discovery. In this study, we generated more than 40 million reads from samples of spleen and skin tissue using the Illumina paired-end sequencing technology. De novo assembly yielded 87,297 transcripts with a mean length of 734 base pairs (bp). Based on the sequence similarities, searching with known proteins, 38,916 genes were identified. Gene enrichment analysis determined that 981 transcripts were assigned to the immune system. Tissue-specific expression analysis indicated that 443 of transcripts were specifically expressed in the spleen and skin. Among these transcripts, 147 transcripts were found to be involved in immune responses and inflammatory reactions, such as fucolectin, β-defensins and lymphotoxin beta. Eight tissue-specific genes were selected for validation using real time reverse transcription quantitative PCR (qRT-PCR). The results showed that these genes were significantly more expressed in spleen and skin than in other tissues, suggesting that these genes have vital roles in the immune response. This work provides a comprehensive genomic sequence resource for A. davidianus and lays the foundation for future research on the immunologic and disease resistance mechanisms of A. davidianus and other amphibians.

Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes

“…The Transrate score [53] is one of the few metrics available for evaluating the 'quality' of a de novo transcriptome. It is similar to the DETONATE RSEM-EVAL score in that it returns a metric indicating how well the assembly is supported by the read data [13]. It does not directly evaluate the underlying de Bruijn graph data structure used to produce the assembled contigs.…”

“…This type of analysis is ubiquitous across many elds, including: evolutionary developmental biology [2], cancer biology [3], agriculture [4,5], ecological physiology [6,7], and biological oceanography [8]. In recent years, substantial investments have been made in data generation, primary data analysis, and development of downstream applications, such as biomarkers and diagnostic tools [9,10,11,12,13,14,15,16] Methods for de novo RNAseq assembly of the most common short read Illumina sequencing data continue to evolve rapidly, especially for non-model species [17]. At this time, there are several major de novo transcriptome assembly software tools available to choose from, including Trinity [18], SOAPdenovo-Trans [19], Trans-ABySS [20], Oases [21], SPAdes [22], IDBAtran [23], and Shannon [24].…”

Data set for transcriptome analysis of the Chinese giant salamander (Andrias davidianus )

“…The unigenes sequences were also aligned based on the KEGG database to analyse metabolic pathways [5] , [6] . Functions categories were carried out based on the COG database [7] , [8] .…”

Sequencing and de novo transcriptome assembly of the Chinese giant salamander ( Andrias davidianus )

“…The Chinese giant salamander is classified as critically endangered by the International Union for Conservation of Nature and Natural Resources (IUCN) Red list of Threatened Species, is a class II on the national list of protected animals in China. Nonetheless, only a limited number of transcriptomes from A. davidianus have been sequenced so far [4], [5], [6], [7], [8]. Meanwhile, the genetic information and gene sequences about the Chinese giant salamander in public databases are also scanty.…”