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

Next-generation technologies for determination of genomics and transcriptomics composition have a wide range of applications. Andrias davidianus, has become an endangered amphibian species of salamander endemic in China. However, there is a lack of the molecular information. In this study, we obtained the RNA-Seq data from a pool of A. davidianus tissue including spleen, liver, muscle, kidney, skin, testis, gut and heart using Illumina HiSeq 2500 platform. A total of 15,398,997,600 bp were obtained, corresponding to 102,659,984 raw reads. A total of 102,659,984 reads were filtered after removing low-quality reads and trimming the adapter sequences. The Trinity program was used to de novo assemble 132,912 unigenes with an average length of 690 bp and N50 of 1263 bp. Unigenes were annotated through number of databases. These transcriptomic data of A. davidianus should open the door to molecular evolution studies based on the entire transcriptome or targeted genes of interest to sequence. The raw data in this study can be available in NCBI SRA database with accession number of SRP099564.

Direct link to deposited data

http://www.ncbi.nlm.nih.gov/sra/SRR5260688

Introduction

The Chinese giant salamander (A. davidianus) belonging to one of the most primitive orders Caudata, family Cryptobranchidae, is the largest extant amphibian species in the world and found only in China [1], [2]. It was called “babyfish” mainly lives in Chinese interior water area. Being crown as a living fossil from 350 million years ago, so it is considered to be a valuable model in edible value, medical value, view value, science value and so on [3]. 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. Here we performed de novo transcriptome assembly for A. davidianus by next-generation sequencing. The obtained transcriptomic data will be useful for further studies of the genomes evolution of A. davidianus may have represented transition steps from aquatic to terrestrial life.

Experimental design, materials and methods

Animal materials

The healthy A. davidianus were obtained from a farm located in Luan chuan (Henan Province, China). Organ tissues including spleen, liver, muscle, kidney, skin, testis, gut and heart collected from three-year-old (3Y) male A. davidianus immediately after dissection, washed in sterile PBS. Animals tissues sample for RNA extraction were snap-frozen in liquid nitrogen and stored at − 80 °C for further analysis. All experiments were performed in strict accordance with the Animal Protection Law of China, and were approved by the Review Committee for the Use of Animal Subjects of Henan University of Science and Technology.

Library construction and sequencing

The A. davidianus were pooled from 8 tissues (spleen, liver, muscle, kidney, skin, testis, gut and heart) and used for total RNAs library construction. RNA-Seq libraries were generated using the TruSeq RNA-Seq Sample Prep kit according to the manufacturer's protocol (Illumina Inc., San Diego, CA). Poly-A RNAs were isolated from total RNA and chemically fragmented. First and second strand cDNA synthesis were followed by end-repair and adenosines were added to the 3′ends. Adapters were ligated to the cDNA and 200 ± 25 bp fragments were gel purified and enriched by PCR. The libraries were quantified using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and run on the Illumina HiSeq2500 platform (Illumina Inc.). Paired-read sequences, 125 nt in length, were collected.

De novo transcriptome assembly and functional annotation

A total of 15,398,997,600 bp were obtained from sequencing libraries. After cleaning and quality checks, the de novo assembly of all sequencing data using the Trinity program. It generated 158,103 all-transcripts with an average length of 810 bp and an N50 of 1659 bp; and 132,912 all unigenes were achieved with an average length of 690 bp and an N50 of 1263 bp (Table 1). Of these, 34,075 were > 1 kb and 21,855 > 1 kb, respectively. We annotated the all 71,443 unigenes to NR, NT, CDD, Swissprot, TrEMBL, Pfam, KEGG, KOG and GO databases. A microsatellite program MISA was also used to identify and localize microsatellite motifs.

Table 1

Summary of A. davidianus assembly statistics.

Index	A. davidianus transcript	A. davidianus unigenes
All number	158,103	132,912
Length ≧ 500 bp	59,715	42,327
Length ≧ 1000 bp	34,075	21,855
N50 length	1659 bp	1263 bp
N90 length	285 bp	262 bp
Max length	16,067 bp	16,067 bp
Minor length	201 bp	201 bp
All length	128,175,999 bp	91,713,308 bp
Mean length	810 bp	690 bp

Conflict of interest

The authors declare that they have no competing interests.

Specifications
Organism/cell line/tissue	A. davidianus/spleen, liver, muscle, kidney, skin, testis, gut and heart
Sex	Male
Sequencer or array type	Illumina HiSeq2500
Data format	Raw data: FASTQ files, analyzed data: txt files
Experimental factors	De novo transcriptome assembly of A. davidianus
Experimental features	Freshly and healthy collected spleen, liver, muscle, kidney, skin, testis, gut and heart were pooled for total RNA extraction, sequencing, de novo transcriptome assembly and annotation
Consent	Full consent
Sample source location	Luoyang, Henan, China