DATA NOTE Open Access
High-quality genome assembly of channel
catfish, Ictalurus punctatus
Xiaohui Chen
1,2†
, Liqiang Zhong
1,2†
, Chao Bian
3†
, Pao Xu
4†
, Ying Qiu
3†
, Xinxin You
3†
, Shiyong Zhang
1,2
, Yu Huang
3
,
Jia Li
3
, Minghua Wang
1,2
, Qin Qin
1,2
, Xiaohua Zhu
1,2
, Chao Peng
3
, Alex Wong
5
, Zhifei Zhu
6,7
, Min Wang
3,6,7
,
Ruobo Gu
4,6
, Junmin Xu
3,6,7*
, Qiong Shi
3,6,7,8,9*
and Wenji Bian
1,2*
Abstract
Background: The channel catfish (Ictalurus punctatus), a species native to North America, is one of the most
important commercial freshwater fish in the world, especially in the United States’ aquaculture industry. Since its
introduction into China in 1984, both cultivation area and yield of this species have been dramatically increased
such that China is now the leading producer of channel catfish. To aid genomic research in this species, data sets
such as genetic linkage groups, long-insert libraries, physical maps, bacterial artificial clones (BAC) end sequences
(BES), transcriptome assemblies, and reference genome sequenc es have been generated. Here, using diverse
assembly methods, we provide a comparable high-quality genome assembly for a channel catfish from a breeding
stock inbred in China for more than three generations, which was originally imported to China from North America.
Findings: Approximately 201.6 gigabases (Gb) of genome reads were sequenced by the Illumina HiSeq 2000
platform. Subsequently, we generated high quality, cost-effective and easily assembled sequences of the channel
catfish genome with a scaffold N50 of 7.2 Mb and 95.6 % completeness. We also predicted that the channel catfish
genome contains 21,556 protein-coding genes and 275.3 Mb (megabase pairs) of repetitive sequences.
Conclusions: We report a high-quality genome assembly of the channel catfish, which is comparable to a recent
report of the “Coco” channel catfish. These generated genome data could be used as an initial platform for
molecular breeding to obtain novel catfish varieties using genomic approaches.
Keywords: Channel catfish, Whole genome sequencing, Assembly, Gene prediction, Repetitive sequence
Data description
Library construction, read sequencing and filtering
To generate genome sequence data, genomic DNA from
mixed tissues (including muscle and skin) of channel
catfish was extracted from a chosen individual cultured at
a local base of the Freshwater Fisheries Research Institute
(Jiangsu Province, Nanjing, China) using Qiagen Geno-
micTip100 (Qiagen, Hilden, DE) as per standard proto-
cols. Isolated genomic DNA was subsequently used to
construct short-insert libraries (250, 500 and 800 bp) and
long-insert libraries (2, 5, 10 and 20 kb) with the standard
protocol provided by Illumina (San Diego, USA). Paired-
end sequencing was performed using the Illumina HiSeq
2000 platform to generate 125-bp reads using a whole
genome shotgun sequencing (WGS) strategy [1].
To improve the quality of sequenced reads, we
trimmed 4 bases with edges from the reads of short-
insert libraries and long-insert libraries, discarded dupli-
cated reads from the long-insert libraries, and removed
reads containing 10 or more Ns and low-qua lity bases.
Finally, a total of 201.6-Gb clean reads were generated
for further genome assembly.
Genome assembly and quality assessments
At first, we estimated the channel catfish genome size
using k-mer analysis [2] with the formula : G = N*(L −
17 + 1)/K_depth, where N is the total number of
* Correspondence:
xujunmin@genomics.cn; shiqiong@genomics.cn; js6060@sina.com
†
Equal contributors
3
Shenzhen Key Laboratory of Marine Genomics, Guangdong Provincial Key
Lab of Molecular Breeding in Marine Economic Animals, Shenzhen 518083,
China
1
Freshwater Fisheries Research Institute of Jiangsu Province, Nanjing 210017,
China
Full list of author information is available at the end of the article
© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Chen et al. GigaScience (2016) 5:39
DOI 10.1186/s13742-016-0142-5
reads, and K_depth indicates the freque ncy of reads
occurring more frequently than the others. The ca lcu-
lated genom e size is 0.8 39 Gb, which is shorter than
that (1 Gb) from a 2016 report of an American-native
channel catfish [3].
Simultaneously, we employed SOAPdenovo2 (version
2.04.4) software [4] with optimized parameters (pregraph
−K27−d 1; contig −M 1; scaff −F −b 1.5 −p 16) to link
sequenced reads to contigs and original scaffolds. All
reads were then aligned onto the contigs for scaffold
construction by utilizing long-insert paired-end informa-
tion, which was subsequently supplied to link contigs to
scaffolds in a step-wise manner. Gaps were closed using
approximately 480 million of Illumina paired-end reads
generated from the three libraries with insert sizes of
250, 500 and 800 bp as the input for GapCloser (v1.12-
r6, default parameters and −p set to 25) [2]. A final gen-
ome assembly of 0.845 Gb in length was obtained
(Table 1), which is slightly shorter than that (0.942 Gb)
of a recently reported a American-native channel catfish
genome [3]. The calculated contig N50 was 48.5 kilo-
bases (kb), and the scaffold N50 was 7.2 Mb (Table 1).
These values are also comparable to those in [3] (see de-
tails in Table 2).
Two typical methods were then used to assess the
quality and completeness of the generated assembly.
First, transcriptome evaluation was used to assess the
completeness of gene regions in the genome assembly.
We carried out de novo a ssembly of the RNA sequences
of skin and muscle tissues using Trinity software [5].
The assembled fragments were then aligned to the
genome assembly with BLAT [6] (E-value = 10e-6, iden-
tity = 90 % and coverage >90 %). Our results indicate
that the catfish genome assembly covered more than
90 % of gene-coding regions. Subsequently, Core
Eukaryotic Genes Mapping Approach (CEGMA) soft-
ware (version 2.3) [7] was employed with 248 conserved
core eukaryotic genes (CEGs) to assess the gene space
completeness within the generated genome assembly.
These results demonstrate that the genome assembly
covered more than 95 % of the CEG sequences, suggest-
ing a high level of completeness.
Transcriptome sequencing
Total RNA was extracted from muscle and skin tissues
of a channel catfish (the same individual used for the
above-mentioned genome sequencing) using TRIzol re-
agent (Invitrogen, USA). After purification using RNeasy
Animal Mini Kit (Qiagen, USA), equal amounts of total
RNA from each tissue were subjected to transcriptome
sequencing (RNA-seq) on the HiSeq 2000 platform.
Genome annotation
Repeat annotation
Firstly, RepeatModeller (version 1.04) and LTR_FINDER
[8] were used to build a de novo repeat library with de-
fault parameters. Subsequently, RepeatMasker [9] (ver-
sion 3.2.9) was utilized to map our sequences against the
Repbase [10] transposable element ( TE) library (vers ion
14.04) and the de novo repeat library, so as to search for
known and novel TEs. Next, we annotated tandem re-
peats using Tandem Repeat Finder [11] (version 4.04)
with core parameters set as “Match = 2, Mismatch = 7,
Delta = 7, PM = 80, PI = 10, Minscore = 50, and Max-
Perid = 2000”. Furthermore, TE-relevant proteins were
identified in our assembly using RepeatProteinMask
software [9] (version 3.2.2). These identified repeat se-
quences accounted for 32.56 % of the channel catfish
genome, of which the single largest class of TEs (repre-
senting 9.35 % of the whole genome) was the Tc1-
mariner family.
Annotations of gene structure and function
The channel catfish genome assembly was annotated
using three independent approaches: homology, de novo
and RNA-seq annotations. For homology annotation, the
protein sequences from zebrafish, Japanese fugu, spotted
green pufferfish, Japanese medaka (Ensembl release 75),
blue spotted mudskipper [1] and golden arowana [12]
were mapped on the channel catfish genome using
TblastN with e-value ≤ 1E-5. Genewise 2.2.0 software
[13] was then employed to predict the potential gene
structures of all alignments. Short genes (with fewer
than 150 bp) and prematurely terminated or frame-
shifted genes were discarded. Next, de novo annotation
was used to annotate the gene structure from the gen-
ome assembly. We randomly selected 1000 complete
genes from the homology annotation set to train the pa-
rameters for AUGUSTUS 2.5 [14]. Simultaneously, all
Table 1 Catfish genome assembly and annotation statistics
Genome assembly
Contig N50 size (kb) 48.5
Contig number (>100 bp) 66,332
Scaffold N50 size (Mb) 7.2
Scaffold number (>100 bp) 31,979
Total length (Mb) 845.4
Genome coverage (X) 201.6
Longest scaffold (bp) 26,612,498
Genome annotation
Protein-coding gene number 21,556
Mean transcript length (kb) 16.1
Mean exons per gene 8.7
Mean exon length (bp) 190.2
Mean intron length (bp) 1872.4
Chen et al. GigaScience (2016) 5:39 Page 2 of 4
repetitive regions were replaced in the channel catfish
genome with ‘N’ to decline the ratio of pseudogene an-
notations. Subsequently, we utilized AUGUSTUS 2.5
and GENSCAN 1.0 [15] for de novo prediction of
repeat-masked genome sequences. The filtered processes
performed on the de novo annotation were the same as
those used for homology prediction. Simultaneously, the
RNA-seq annotation pipeline was also used to detect
gene regions. We employed Tophat 1.2 software [16] to
map the RNA reads extracted from the skin and muscle
transcriptomes onto the channel catfish genome se-
quences. We then sorted and integrated Tophat align-
ments, and used Cufflink software [17] to analyze
potential gene structures. Results from all three of the
above-mentioned annotation pipelines were merged to
produce a comprehensive and non-redundant gene set
using GLEAN [18]. This gene set contained 21,556 genes
with an average of 8.7 exons per gene (Table 1). Because
different annotation pipelines were applied, the total
gene number predicted here is lower than the 26,661 re-
ported in the American-native channel catfish genome
[3]. The Cuffdiff package [17] of Cufflink software (ver-
sion 2.0.2.Linux_×86_64) with core parameters (−FDR
0.05 –geometric-norm TRUE –compatible-hits-norm
TRUE) was utilized to calculate expression levels accord-
ing to the GLEAN gene set and Tophat alignmen ts.
About 93.4 % of genes were pred icted from at least two
types of evidence, and approximate 78 % of the genes
showed expression activity (fragments per kilobase of
exon model per million mapped reads >0) in the skin
and muscle tissues.
Simultaneously, all protein sequences from GLEAN
results were mapped to SwissProt and TrEMBL [19]
(UniProt release 2011.06) databases using BlastP [20]
with an E-value ≤ 1e-5 to find the best hit for each pro-
tein. We also used InterProScan 4.7 software [21] to
align the protein sequences against public databases, in-
cluding Pfam [22], PRINTS [23], ProDom [24] and
SMART [25], to examine the known motifs and domains
in our sequences. Over 94.5 % of these predicted genes
possessed at least one related functional assignment
from other public databases (SwissProt [19], Interpro
[21], TrEMBL and KEGG [26]). In addition, the gene
structures (including exon length, intron regions and
mRNAs) and exon number distributions (Table 1) were
predicted to be similar to other representative teleost
species such as zebrafish and medaka.
Conclusion
We generated a channel catfish genome assemb ly with
high quality and comparable structures to other pub-
lished fish genomes, especially the Coco catfish genome
[3]. This new assembly is a valuable resource and refer-
ence for further construction of high-density genetic
linkage maps and identification of quantitative trait loci
for molecular breeding of catfishes.
Availability of supporting data
Supporting data are available in the GigaDB database
[27]. Raw whole genome sequencing and transcriptome
data are deposited in the SRA under bioproject number
PRJNA319455.
Abbreviations
BAC, bacterial artificial clone; BES, BAC end sequences/sequencing; CEG, core
eukaryotic genes; CEGMA, core eukaryotic genes mapping approach; Gb,
gigabases; kb, kilobases; Mb, megabases; TE, transposable element; WGS,
whole genome shotgun
Table 2 Comparison of genome assembly in sequenced fishes
Species Sequencing platform (Mb) Assembled genome size (Mb) scaffold N50 (kb) contig N50 (kb)
catfish (BGI) Illumina 845 7248 48.5
catfish (Liu’s study [1]) Illumina, Pacbio 942 7726 77.2
zebrafish Illumina, Sanger 1412 1551 25.0
Atlantic herring Illumina 808 1840 21.3
greenpuffer Sanger 342 100 16.0
medaka Sanger 700 1410 9.8
stickleback Sanger, Illumina 463 10,800 83.2
fugu Sanger 332 unknown 16.5
cod 454 753 459 2.8
platyfish 454, Illumina 669 1102 21.0
lamprey 454, Illumina 816 173 unknown
lancelets Illumina 520 unknown unknown
tuna 454, Illumina 800 136 7.6
mudskipper Illumina 983 2309 20.0
Chen et al. GigaScience (2016) 5:39 Page 3 of 4
Funding
This study was supported by the National Key Technology R&D Program of
China (No. 2012BAD26B03), Fund for Independent Innovation of Agricultural
Science and Technology of Jiangsu Province (No. CX(15)1013), Human
Resources and Social Security of Jiangsu Province (No. 2014-NY-008), Three-
Side Innovation Projects for Aquaculture in Jiangsu Province (No. Y2014-25
&Y2015-12), Shenzhen Special Program for Future Industrial Development
(No. JSGG20141020113728803), and Zhenjiang Leading Talent Program for
Innovation and Entrepreneurship.
Authors’ contributions
XC, QS, CB, JX and WB conceived the project. LZ, YH, SZ, MHW, QQ, XY, CP, AW,
ZZ, MW and RG collected the samples and extracted the genomic DNA. CB, YQ,
JL and YH performed the genome assembly and data analysis. CB, QS, XC, LZ,
XP, XZ and WB wrote the paper and all authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Freshwater Fisheries Research Institute of Jiangsu Province, Nanjing 210017,
China.
2
The Jiangsu Provincial Platform for Conservation and Utilization of
Agricultural Germplasm, Nanjing 210017, China.
3
Shenzhen Key Laboratory of
Marine Genomics, Guangdong Provincial Key Lab of Molecular Breeding in
Marine Economic Animals, Shenzhen 518083, China.
4
Freshwater Fisheries
Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China.
5
BGI-Hong Kong, Hong Kong 999077, China.
6
BGI Zhenjiang Institute of
Hydrobiology, Zhenjiang 212000, China.
7
BGI Zhenjiang Fisheries Science and
Technology Industrial Co. Ltd, Zhenjiang 212000, China.
8
Laboratory of
Aquatic Genomics, College of Ecology and Evolution, School of Life Sciences,
Sun Yat-Sen University, Guangzhou 510275, China.
9
Center for Marine
Research, College of Life Sciences and Oceanography, Shenzhen University,
Shenzhen 518060, China.
Received: 20 May 2016 Accepted: 3 August 2016
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