A combined strategy involving Sanger and 454 pyrosequencing increases genomic resources to aid in the management of reproduction, disease control and genetic selection in the turbot (Scophthalmus maximus)
© Ribas et al.; licensee BioMed Central Ltd. 2013
Received: 12 October 2012
Accepted: 27 February 2013
Published: 15 March 2013
Genomic resources for plant and animal species that are under exploitation primarily for human consumption are increasingly important, among other things, for understanding physiological processes and for establishing adequate genetic selection programs. Current available techniques for high-throughput sequencing have been implemented in a number of species, including fish, to obtain a proper description of the transcriptome. The objective of this study was to generate a comprehensive transcriptomic database in turbot, a highly priced farmed fish species in Europe, with potential expansion to other areas of the world, for which there are unsolved production bottlenecks, to understand better reproductive- and immune-related functions. This information is essential to implement marker assisted selection programs useful for the turbot industry.
Expressed sequence tags were generated by Sanger sequencing of cDNA libraries from different immune-related tissues after several parasitic challenges. The resulting database (“Turbot 2 database”) was enlarged with sequences generated from a 454 sequencing run of brain-hypophysis-gonadal axis-derived RNA obtained from turbot at different development stages. The assembly of Sanger and 454 sequences generated 52,427 consensus sequences (“Turbot 3 database”), of which 23,661 were successfully annotated. A total of 1,410 sequences were confirmed to be related to reproduction and key genes involved in sex differentiation and maturation were identified for the first time in turbot (AR, AMH, SRY-related genes, CYP19A, ZPGs, STAR FSHR, etc.). Similarly, 2,241 sequences were related to the immune system and several novel key immune genes were identified (BCL, TRAF, NCK, CD28 and TOLLIP, among others). The number of genes of many relevant reproduction- and immune-related pathways present in the database was 50–90% of the total gene count of each pathway. In addition, 1,237 microsatellites and 7,362 single nucleotide polymorphisms (SNPs) were also compiled. Further, 2,976 putative natural antisense transcripts (NATs) including microRNAs were also identified.
The combined sequencing strategies employed here significantly increased the turbot genomic resources available, including 34,400 novel sequences. The generated database contains a larger number of genes relevant for reproduction- and immune-associated studies, with an excellent coverage of most genes present in many relevant physiological pathways. This database also allowed the identification of many microsatellites and SNP markers that will be very useful for population and genome screening and a valuable aid in marker assisted selection programs.
KeywordsTurbot NGS Database Transcriptome Reproduction Immune Genetic Markers SNP Microsatellite Microarray Natural Antisense Transcripts MicroRNA
The turbot (Scophthalmus maximus) is a flatfish with increasing commercial relevance in Europe with a current annual production of ~10,000 tones  with an increasing consumer demand worldwide. Thus, turbot production significantly increased in Northern China during the last decade. However, fish disease outbreaks collapsed its production in 2006, with economic losses estimated to amount several hundred million Euros [2, 3].
It seems clear that one of the major concerns for turbot aquaculture is disease control. Intensive culture conditions in fish farms favors the proliferation of pathogens and the consequent economic losses associated with disease outbreaks. Hence, a comprehensive knowledge of the immune system of commercially important fish species is required . The immune-prophylactic control of fish diseases through vaccination, probiotics and immunostimulation has been undertaken since long ago [5–7], whereas genetic programs on disease resistance, specifically in turbot, clearly require further investigation. Obtaining resistant broodstock is an appealing solution to control diseases in front of the economic cost of vaccines, treatments and the possible generation of resistances against antibiotics.
Another major concern for the aquaculture industry is fish reproduction. Like in other vertebrates, reproduction in turbot is controlled by the brain-pituitary-gonad axis, which integrates environmental signals and controls the production and secretion of the major hormones involved in controlling the reproductive cycle, including the onset of puberty [8, 9]. Furthermore, turbot exhibits one of the largest cases of sexual dimorphism for growth rate in favor of females among aquacultured species . Therefore, there is an interest in the turbot aquaculture industry to produce stocks with as many females as possible in order to increase biomass. Gonad development is a complex biological process in which an undifferentiated bipotential gonad is transformed into either a testis or an ovary [11, 12] according to sex determination and differentiation [11, 13]. External factors such as temperature, pH or social behavior can directly influence gonadal development in some fish [14, 15] and, consequently, affect sex ratio. Understanding the process of gonadal development can greatly aid in the control of sex ratios in finfish aquaculture. However, in turbot there is a lack of information of genes involved in reproduction and their interactions. The induction of gynogenesis suggested a XX/XY system of sex determination , but later studies involving the analysis of progenies from sex-reversed parents revealed a ZW/ZZ system . Linkage maps were developed [18–21] and led to the identification of the major sex-determining region  and facilitated the characterization and mapping of sex-associated markers [23, 24], although the sex determining gene(s) is (are) still unknown.
Increase of the genomic resources for the turbot ( S. maximus ) with the successive databases
No. of sequences (reads)
No. unique sequences
(contigs + singletons)
Liver, head kidney, spleen (Aeromonas and Philasterides infection)
ABI3730 cDNA library
Pardo et al., 2008 
(1,073 + 2,409)
ABI3730 Microsatellite- enriched DNA library
Pardo et al. 2006 
Liver, kidney and gills (nodavirus infection and stimulation
ABI3730 cDNA library
Park et al., 2009 
Liver, head kidney, spleen, pyloric caeca and thymus (Enteromyxum infection)
ABI3730 cDNA library
Ribas et al., present work
(1,827 + 4,343)
454 Roche Titanium
52,427 + 176,451
Ribas et al., present work
Next Generation Sequencing (NGS) strategies have positively affected genetics research over the last few years and their advantages have been applied to many research fields. 454 FLX Titanium is a massive pyrosequencing strategy which generates medium-size single reads uncovering large amounts of DNA sequences providing much deeper sequencing coverage than it is possible with conventional Sanger sequencing . Sequencing small subsets of the genome such as the transcriptome is an attractive alternative for gene discovery in species whose genome is still not available, and fish are not an exception. For example, in guppy (Poecilia reticulata) the sequencing of a total of 336 megabases (Mb) produced the first reference transcriptome for this fish species . In the self-fertilizing hermaphroditic mangrove Rivulus, Kryptolebias marmoratus, the identification of more than 150,000 sequences provided the first insights on the mechanisms underlying the response to environmental stresses and chemical toxicities ; and in the gilthead sea bream (Sparus aurata), the fast skeletal muscle transcriptome was described . In particular, the reproductive system of the lake sturgeon (Acipenser fulvescens) has also been studied by resorting to modern pyrosequencing and it has been useful for the discovery and evaluation of candidate sex-determining genes and xenobiotic-responsive genes in the gonads [44, 45].
Another approach to improve the aquaculture production is based on the application of molecular markers such as microsatellites or simple sequence repeats (SSRs) and SNPs. These markers are the basis for genetic mapping and comparative genomic analysis, which are in turn used for detection of quantitative trait loci (QTL) and for marker assisted selection (MAS) programs [46, 47]. Several types of genetic markers have been developed and investigated in turbot  and many of them have already been mapped [18–21]. Recently, a genome scan for sex-determination  and resistance/survival to A. salmonicida and P. dicentrarchi using the genetic map identified several consistent QTLs and associated markers in turbot, which suggests the existence of genetic factors underlying these characters and supports their application in genetic breeding strategies. The advents of new high-throughput sequencing technologies, which produce extensive sequence data, are providing new opportunities to increase the amount of molecular markers, as demonstrated in the sturgeon, where hundreds of SNPs were discovered .
Overall, the improvement of the turbot aquaculture industry by selecting, on one hand, the most resistant broodstock and, on the other hand, female-biased batches is a priority challenge. The purpose of this study was to increase turbot database information for genes related to the immune and reproductive systems by creating a powerful tool for genomic research in this species. The turbot database was updated with genes obtained both by Sanger sequencing from immune-related tissues after challenges with the myxozoan parasite E. scophthalmi and by a 454 FLX Titanium run from gonad and brain-hypophysis at different stages of development. Description and comparison of the two sequencing strategies, annotation procedures, and construction of a larger database, the support for microsatellites and SNP discovery, and for designing a pilot-microarray platform, are presented.
Results and discussion
The increase of known immune-related genes in turbot by Sanger sequencing
The progression in the construction of the turbot database is summarized in Table 1. First, the Turbot 1 database was created from almost ten thousand high-quality EST sequences from three cDNA libraries of three immune relevant organs (liver, head kidney and spleen) generated from turbot infected with A. salmonicida subspecies salmonicida and P. dicentrarchi, as well as from non-infected fish . The Turbot 2 database included several resource sequences: i) 1,371 sequences from seven microsatellite-enriched DNA libraries from muscle tissues ; ii) 3,339 ESTs available in public databases , which were loaded on the turbot database and clustered with the set of the existing EST; and iii) Sanger sequencing data from two new cDNA libraries generated from several immune tissues (liver, head kidney, spleen, pyloric caeca and thymus) after challenging with the myxosporean parasite E. scophthalmi produced a total of 3,043 sequences (see Methods). Together, Sanger-based sequencing generated 17,626 sequences with an average length of 501 base pair (bp), constituting the Turbot 2 database. The assembly of all these available data consisted of 6,170 putative transcripts of which 1,827 were contigs and 4,343 singletons. A high level of redundancy was found (75.6%), which is usually observed when non-normalized cDNA libraries are used , but it constitutes an appropriate approach to obtain a first picture of the immune response .
A total of 6,053 out of the 6,170 unique sequences (98.1%) in Turbot 2 database displayed significant matches with sequences available in public databases with E-values equal or less than 1,00E-5 (6,049 with BLASTN and 2,116 with BLASTX). Gene Ontology (GO) annotation classified sequences as follows: 586 in Biological Process (BP), 472 in Cellular Component (CC) and 692 in Molecular Functions (MF).
454 pyrosequencing of the turbot brain-hypophysis-gonad axis transcriptome
Summary statistics of ( S . maximus ) 454-pyrosequencing
Total reads (raw wells)
High quality reads (filtered)
Total megabases (Mb)
Average read length (bp)
N50 read length (bp)
Number of contigs
Number of contigs > 500 bp
Number of singletons
Total consensus length (Mb)
Average contig coverage
Mean contig length (bp)
Median contig length (bp)
Mode contig length (bp)
N25 contig length (bp)
N50 contig length (bp)
N75 contig length (bp)
List of the top 20 longest contigs originated from the 454 run of brain-hypophysis-gonad axis tissues of turbot ( S . maximus )
Average coverage per nucleotide
Cytochrome c oxidase subunit 3
Adrenodoxin-like protein mitochondrial
SSRU rRNA Cavia porcellus
Ubiquitin carboxyl-terminal hydrolase
Novel protein (Zgc:55794)
Novel protein similar to WDR44
Cell division cycle
M-phase phosphoprotein 10
NADH dehydrogenase subunit 5
Suppressor of tumorigenicity 7 protein
60S ribosomal protein
NF-kappaB repressing factor
WD repeat domain phosphoinositide
List of the top 20 deepest contigs originated from the 454 run of brain-hypophysis-gonad axis tissues of turbot ( S . maximus )
Average coverage per nucleotide
40S ribosomal protein S9
Nucleolar protein family A3
Similar to ribosomal protein S7
NADH dehydrogenase 1 beta
Chromobox protein homolog 3
Ribosomal protein L7
Fatty acid binding protein 11a
General transcription factor IIIA
Ribosomal protein S11
Histone deacetylase complex
40S ribosomal protein S27a
Ribosomal protein L12
60S ribosomal protein L13
Epididymal secretory protein E1
About half of the contigs obtained in the 454 run were successfully annotated and classified into Gene Ontology categories. More precisely, contigs exclusively obtained by the 454 run were functionally classified in the BP (8,390), CC (7,081) and MF (10,026) categories.
Creation of the turbot 3 database
The sequencing strategies used, i.e. traditional Sanger and high throughput 454, yielded a high amount of transcriptomic sequences both from immune and reproductive systems in turbot. With all the information generated, a new Turbot 3 database was created and stored in a web-based portal for exploitation, first by the consortium participating in this project and then publically once the project is finished by the end of 2013. Cap3 software was used to assemble the sequences coming from all Sanger-based libraries (17,626) and the contigs from 454-pyrosequencing (65,472), yielding 52,427 unique sequences (47,134 only-454, 2,904 mixed Sanger-454 and 2,389 only-Sanger), thus reducing redundancy among sequences (Table 1). The number of sequences generated in one single pyrosequencing run (65,472) was almost four times higher than the six libraries sequenced by Sanger together (17,626). When comparing to public turbot resources, our strategy allowed increasing by 34,400 the number of novel sequences identified for the first time in turbot.
Annotation of the turbot 3 database
Identification of genes related to the immune response
The knowledge of the immune system of fish has greatly increased recently. However, there are still many fish diseases which produce important losses to industry because still there is no an effective strategy for their control, including vaccines. The immune system of fish is composed of non-specific and specific immune defenses, being the first more important than in higher vertebrates [56, 57]. Examples of innate immunity include anatomic barriers, mechanical removal of pathogens, bacterial antagonism, pattern-recognition receptors, antigen-nonspecific defense compounds, the complement pathway, phagocytosis, and inflammation . In the present study, the main organs of the immune system of fish such as head kidney (equivalent to the bone marrow of mammals), spleen and thymus (often neglected in fish immune transcriptomics) were included. In addition, other organs such as the liver, a multifunctional organ with innate immune functions in mammals  and poorly studied in fish, and the pyloric caeca, the target organ of the myxozoan parasite, which also plays a role in immunity , were included as well.
Selection of some of the novel relevant immune-related genes identified in the Turbot 3 database
DNA fragmentation factor, 40kDa, beta polypeptide
GO:0006917 GO:0006309 GO:0030263
Tumor necrosis factor receptor-associated factor 2
GO:0045087 GO:0008624 GO:0050870 GO:0042981
Interleukin 1 receptor activated kinase 1
GO:0045087 GO:0008063 GO:0006916
JNK1/MAPK8-associated membrane protein
GO:0045087 GO:0006954 GO:0045321
TNF receptor-associated factor 6
GO:0045087 GO:0006915 GO:0008063 GO:0050852
FYN oncogene related to SRC, FGR, YES
Cytoplasmic protein 1
GO:0042110 GO:0050852 GO:0042102
Cytoplasmic protein NCK2
Disks large homolog 1
Mitogen-activated protein kinase 8
GO:0031295 GO:0000165 GO:0000186
T-cell-specific surface glycoprotein CD28
GO:0031295 GO:0006959 GO:0008624 GO:0042102 GO:0045768 GO:0002863 GO:0045086 GO:0045066
GRB2-related adaptor protein 2
GO:0031295 GO:0050852 GO:0007265 GO:0007267
Growth factor receptor-bound protein 2
GO:0031295 GO:0050900 GO:2000379 GO:0030168 GO:0007265
Several immune related pathways were also identified in the Turbot 3 database. Chemokine signaling is an important immune pathway due to the fundamental role of chemokines in providing directional cues for the trafficking of leukocytes to sites of inflammation but also it has been implicated in dendritic cell maturation, macrophage activation, neutrophil degranulation, B cell antibody class switching, and T cell activation. The data infers that chemokines influence both the innate and acquired phase of an immune response to host insults. Thus, the protein richness of this pathway in the Turbot 3 database was described (see Additional file 2). Most members intervening in this pathway were identified showing the usefulness of the Turbot 3 database for gene discovery.
Identification of genes related to reproduction
To date, fish gonad-related ESTs are poorly represented in public databases. A first attempt to identify genes related to gonad development in male and female turbot was carried out by cDNA-AFLP technology and several specific sequences could be identified . However, the amount of information presently available is still scarce and thus a small number of sex-specific sequences have been identified. Here, the use of the 454 FLX Titanium sequencing allowed obtaining a large number of gene sequences (65,472 contigs) and their subsequent assembly and gene annotation led to the identification of a total of 1,410 annotated sequences related to reproductive function. This means that sequences corresponding to many genes of the brain-hypophysis-gonad axis, expressed first during the process of sex differentiation and then during gonadal maturation, have been identified (Additional file 3). Functional annotation terms classified all those sequences in a total of 8,425 GO terms. This is the first time that sex-related genes have been massively identified towards understanding gonad development and maturation in the turbot.
Selection of some of the novel relevant reproductive-related genes identified in the Turbot 3 database
Androgen receptor alpha
GO:0005634 GO:0003707 GO:0003700 GO:0006355
Cytochrome P450 aromatase
GO:0009055 GO:0020037 GO:0005506 GO:0004497
Follicle stimulating hormone receptor
Gonadal soma derived factor
Growth differentiation factor 9
Meiotic nuclear division protein 1 homolog
Mitotic arrest deficient 2
Müllerian inihibiting substance
Sex hormone binding globulin
SRY-box containing gene 6
SRY-box containing gene 9
Spermatogenesis associated 13
GO:0005089 GO:0005622 GO:0035023
StAR-related lipid transfer protein 5
GO:0006694 GO:0015485 GO:0017127
StAR-related lipid transfer protein 7
GO:0009055 GO:0020037 GO:0005506 GO:0004497
Zona pellucida glycoproteins
Zygote arrest protein 1
Another group of identified genes in turbot is involved in ovarian development. This is the case of the cytochrome P450 aromatase (CYP19A), Zona pellucida glycoprotein (ZPG) or the Zygote arrest protein. CYP19A is a key enzyme in the hormonal steroidogenic pathway that mediates the conversion of androgens into estrogens , with two isoforms with specific regulation and tissue distribution [72, 73] and it has been cloned in several cultured fish species like European sea bass . Higher expression of CYP19A is found in female gonads when compared to male gonads from early development and recently it has been shown that different methylation levels of its promoter are related to temperature during the thermal sensitive period . ZPGs are glycoproteins found in the fish chorion, which mediate species-specific sperm binding . ZPGs are encoded by multiple gene families and here several of them have been identified (ZPG2, 3, 4). The steroidogenic acute regulatory protein (STAR) and similar proteins containing STAR-related lipid transfer (START) are responsible for the synthesis of sex steroids and other hormones like cortisol in response to specific stimuli . Here, we were able to identify two different START genes, START5 and START 7.
Gonadotropins (GtHs) control the complex endocrine system that regulates gonadal growth, sexual development and reproductive function, and are secreted by the hypophysis . Three forms of GnRH in the brain and pituitary of the turbot have been identified so far . One of the main gonadotropins in vertebrates, as well in fish, is the follicle-stimulating hormone (FSH). Its receptor, FSHR, is found in male and female gonads and although cloned in other cultured fishes such as the sea bass , here is it identified for the first time in turbot.
Not only genes expressed in somatic cells but also genes expressed in the germ cell line were present in the Turbot 3 database. VASA plays an important role in germ cell determination and development and is an essential factor for primordial germ cell formation and migration to the germinal ridge . In fish, VASA was first cloned in zebrafish  and later in rainbow trout  but also in turbot (JX235364).
Representation of reproductive pathways with more than 50% of coverage in the Turbot 3 database
No. of genes in the pathway
Nº of genes in the database
mTOR signaling pathway
ErbB signaling pathway
Progesterone-mediated oocyte maturation
GnRH signaling pathway
Insulin signaling pathway
Wnt signaling pathway
Notch signaling pathway
Overall, our results show that the approach followed was successful since most of the well-known reproduction-related genes found in other species have been also identified in turbot essentially at once.
An important emerging application of high-throughput 454 sequencing is the identification of molecular markers from genomic DNA. In fact, recent studies have identified 26 polymorphic microsatellite by pyrosequencing in an endangered fish species of China  and 21 microsatellites loci from the threatened freshwater Yarra pygmy perch (Nannoperca obscura) . However, few studies have been conducted to search for cDNA-associated microsatellites, like those identified in the Atlantic herring (Clupea harengus) , despite the potential for targeting candidate genes .
Frequency distribution of the new SSRs by motif length in the Turbot 3 database
Repeat unit number
SSR motif length
% excluding 4 and 5 repetitions
Summary statistics of SNPs in the Turbot 3 database
Total number SNPs
Total contigs with SNPs
with 4 sequences
with 5-10 sequences
with 11-20 sequences
with 21-30 sequences
with 31-50 sequences
with > 50 sequences
Total number of transitions
Total number of transversions
Total number of indels
The large amount of potential molecular markers found in this study will enable more detailed population and applied genomic studies. Since these new markers are linked to genes, they will be useful as Type I markers for population genomics screening in this species and for comparative mapping and fish evolutionary studies .
Pilot microarray and identification of natural antisense transcripts
To date, several custom microarrays have been designed in several non-model fish species. Examples exist in rainbow trout  gilthead sea bream , European sea bass , Atlantic salmon (Salmo salar) , common carp (Cyprinus carpio)  or Senegalese sole (Solea senegalensis) , but also in the turbot . In the present study, samples from the reproductive and immune tissues were used to characterize their transcriptome using different sequencing strategies and de novo assembly to identify a large number of genes previously unknown in turbot. The assembled data present in the Turbot 3 database was the basis to construct a pilot microarray towards a new gene-enriched updated version. One of the drawbacks of 454 sequencing technology is that it may produce false annotations of genes [100, 101], and since sequencing is not oriented as in cDNA libraries used for Sanger sequencing, it is not possible to know the DNA sense strand of a gene unless it is confidently annotated. To solve these problems, and in order to identify the most reliable oligos for a definitive turbot microarray, a pilot microarray was developed. In this pilot microarray, oligos were designed both in forward and reverse sequence orientation. In addition, several filtration criteria were followed to analyze microarray data (see Methods). This strategy allows, on one hand, to identify the sense strand of the non-annotated sequences, but also to identify false annotation of genes. On the other hand, this procedure also allows studying the frequency of putative natural antisense transcripts (NATs) in turbot transcriptome .
The importance of NATs, which can regulate eukaryotic gene expression, has emerged in the last decade . A NAT is a single-stranded RNA sequence complementary to messenger RNA and includes various classes of short RNAs including micro RNAs (miRNAs), promoter-associated transcripts and long non-protein-coding RNAs [103, 104]. The amount of NATs in eukaryotic cells remains unclear. It had been reported that over 20% of human transcripts might form sense–antisense pairs , but large-scale cDNA sequencing suggested that antisense transcription is more common than previously thought . Recently, it has been shown that up to 72% of the transcripts had antisense partners in human and mouse transcriptomes [107, 108]. High-throughput sequencing strategies have revealed a plethora of non-protein-coding transcripts from both genic and intergenic regions . Data on miRNAs, one of the short NAT classes, has been already published in rainbow trout  and halibut Hippoglussus hippoglossus. Due to their increasing importance, the study of NATs cannot be longer ignored in transcriptome studies.
Filtration process results for the 47,921 sequences with oligos in forward and reverse orientation
2 nd filtration
Representative sample of miRNAs found in the Turbot 3 database. miRNAs were identified by Blasting Turbot 3 database sequences against the miRBase
Turbot 3 database annotation
miRBase accession name
Early growth response 1
Cricetulus griseus miR-29a stem-loop
Bos taurus miR-6529 stem-loop
Serine/threonine-protein phosphatase 2A
Homo sapiens miR-3160-2 stem-loop
Similar to dynamin 3
Mus musculus miR-204 stem-loop
Similar to Elongation factor 1-gamma
Ciona intestinalis miR-4064 stem-loop
Similar to ORFa
Oryzias latipes miR-133-2 stem-loop
Similarity to transposases
Oryza sativa miR169i stem-loop
Spindle and kinetochore-associated protein 2-like
Anolis carolinensis miR-301a stem-loop
Glycine max miR5769 stem-loop
Unnamed protein product
Homo sapiens miR-1183 stem-loop
This is the first time that the transcriptome of the reproductive and the immune systems of turbot have been widely explored together. Both systems are essential for the survival of individuals and are of primary importance for commercial aquaculture. This study was designed to fill in the gap of genomic resources in turbot and therefore to improve available turbot sequence databases, specifically in genes related to reproduction. The large amount of generated sequences (52,427 putative transcripts) resulted in one of the most complete available databases for flatfish, with more than half of the resources annotated by both gene and functional category. The detailed and focused sequence assembly and gene annotation strategies allowed the identification of several genes involved in the immune and the reproductive systems, being most of them involved in key functions. A large amount of genetic markers was identified, providing new tools for genomic studies. The performance of an informative pilot microarray was assessed and identification of putative miRNAs was possible. Thus, NGS technologies represent an essential tool to increase exponentially genomic resources in non-model species, opening new insights for our understanding of key biological processes and addressing production bottlenecks in their aquaculture.
Animals were treated according to the Directive 2010/63/UE of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for experimentation and other scientific purposes. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Santiago de Compostela (Spain).
Experimental design and samplings
The E. scophthalmi infection trial was performed at the facilities of CETGA (Centro Tecnológico Gallego de Acuicultura; NW Spain). Naïve turbot (8484 recipients = R and 8484 controls = C) from a balanced mixture of five unrelated families with known pedigree, hatched and reared at a commercial fish farm were sent to CETGA facilities and acclimated to experimental conditions for 10 days before the beginning of the experiment. R and C fish were kept in separate tanks (7 tanks for each group) in two separated recirculating systems, with constant water temperature (19–20°C) and fed with commercial dried pellets. R fish were infected by oral intubation with intestinal scrapings containing E. scophthalmi stages obtained from infected turbot, for two consecutive days. C fish were maintained under equivalent conditions as R fish, but intubated with PBS instead. More details on this procedure can be found in a previous work .
The progression of the infection was monitored by sampling both C and R groups at different times post inoculation (p.i.). The prevalence of infection at each sampling point was obtained by detecting positive fish by either PCR or histology in any of the organs examined. At each sampling point, 14 fish from each group were sized, weighed and euthanized by over-exposure to benzocaine (3-aminobenzoic acid ethyl ester, 100 mg L-1, Sigma, St. Louis, MO, USA). The resulting prevalence of infection was 0, 7.1, 28.6, 85.7 and 92.9% at 4, 7, 14, 25 and 34 days p.i, respectively. No C fish was found to be infected.
Samples of spleen, head kidney, thymus, liver and pyloric caeca were rapidly dissected, immediately frozen in liquid nitrogen and stored at −80°C until used for RNA extraction. At each sampling time, samples of each tissue from the different individual fish from each group (C, R) were pooled. The serial times of sampling provided tissues expressing different genes related to immune response from initial until late states of the infection.
RNA isolation, library preparation and sequence analysis
RNA extraction of samples from control and infected fish, cDNA library construction and sequencing were performed as described elsewhere . Briefly, RNA was extracted using TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA). Poly-A mRNA was isolated using the Dynabeads® mRNA Purification Kit (Invitrogen, Carlsbad, CA, USA). The two cDNA libraries (control and infected) were directionally constructed (5′EcoRI, 3′XhoI), with equal amounts of RNA from each tissue at each sampling time, using the ZAP-cDNA Library Construction Kit (Stratagene, La Jolla, CA, USA), except size fractioning that was performed with the SizeSep 400 Spun Columns (GE Healthcare, Uppsala, Sweden). Plasmid DNA was isolated from approximately 4,000 clones from each library using the DirectPrep® 96 Miniprep kit (QIAGEN, Valencia, CA, USA). Plasmid DNA was sequenced following the ABI Prism BigDye™ Teminator v3.1 Cycle Sequencing Kit protocol on an ABI 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA). All clones were sequenced from their 3′ ends using a standard T7 primer to obtain the highest specific gene sequences for oligo-microarray design. Those clones that suffered a systematic drop on sequencing signal after poly-A tails were sequenced from the 5′ end. Basecalling from chromatogram traces was performed by using PHRED [113, 114].
454 pyrosequencing run
Reproductive tissue sampling and RNA extraction
A total of 30 turbot samples were collected from CETGA from a mixture of unrelated genetic families. In order to obtain the widest possible range of expressed transcript sub-sets, tissues were dissected in fish at different stages of gonad development. The number, age and the mean values of biometry (standard length and body weight) for each animal group were the following: undifferentiated animals (n = 5; 90 days post hatch [dph], 5.2 ± 0.6 cm, 2.9 ± 0.9 g); differentiating animals (n = 4; 150 dph, 9.8 ± 1.3 cm, 21.8 ± 9.5 g); male juveniles (n = 4; 400 dph, 20.9 ± 4.2 cm, 195.0 ± 123.0 g); female juveniles (n = 5; 450 dph, 23.4 ± 3.6 cm, 264.1 ± 93.4 g); male broodstock (n = 3; 900 dph, 44.8 ± 3.6 cm, 2,059.0 ± 591.6 g) and female broodstock (n = 3; 900 dph, 47.7 ± 6.4 cm, 2,834.3 ± 1,264.9 g). Brain and hypophysis from broodstock animals were also dissected and rapidly flash frozen in liquid nitrogen. Gonads were fully isolated in adult and juvenile fish and thus gonadal tissue was devoid of any other tissue. However, gonads of sexually differentiating fish contained a bit of attached epithelium. Due to their extremely small size, the isolation of the gonads alone was not feasible and thus samples contained also portions of the surrounding tissues.
RNA was individually extracted by RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Quantity was determined using a Nanodrop spectrophotometer (Nanodrop Technologies, US). The RNA integrity number (RIN) was determined in an Agilent BioAnalizer (Agilent Technologies, US). RNA samples with a RIN > 8.1 were further processed for the sequencing run. A pooled sample was generated by mixing 70% of gonads containing equal amounts of RNA from each individual and 30% of equal amount of RNA from broodstock brains and hypophysis tissues.
cDNA library, normalization and 454 FLX Titanium pyrosequencing
Full-length-enriched double stranded cDNA was synthesized from 1.5 μg of pooled total RNA using the MINT cDNA synthesis kit (Evrogen, Moscow, Russia) according to the manufacturer’s protocol, and was subsequently purified using the QIAquick PCR Purification Kit (Qiagen USA, Valencia, CA). The amplified cDNA was normalized using the Trimmer kit (Evrogen, Moscow, Russia) to minimize differences in representation of transcripts [115, 116]. The single-stranded cDNA fraction was then amplified twice by sequential PCR reactions according to manufacturer’s protocol. Normalized cDNA was purified using the QIAquick PCR Purification Kit (Qiagen USA, Valencia, CA). Normalized cDNA (5 μg) was used to generate a 454 library. cDNA was fractionated into small, 300 to 800 bp fragments and the specific A and B adaptors were ligated to both the 3′ and 5′ ends of the fragments and used for purification, amplification, and sequencing steps. Two and a quarter PTP regions were used for the GS-FLX sequencing run using Titanium chemistry. All reagents and protocols were from Roche 454 Life Sciences, USA. 454 data was processed with Roche’s software, using default settings, to obtain fasta and quality files containing the trimmed sequence of all reads . Contigs with at least 100 bp were recovered. Sequences were de novo assembled into contigs by running Mira v3.2.0rc1 in EST mode. Contigs less than 100 bp were filtered out and the rest was blasted against D. rerio RefSeq protein sequences with est2assembly’s analyse_assembly.pl script  in order to validate the whole process.
Bioinformatic tools were developed to process all sequencing data obtained from both Sanger and 454 FLX Titanium technologies. The starting point of the current work was the Turbot 1 database, which was reported previously . In order to generate the Turbot 2 database sequences of Turbot 1 database (9,873) were clustered with: 3,043 sequences obtained from the E. scophthalmi trial cDNA libraries, 1,371 genomic sequences from enriched DNA libraries  and 3,339 sequences available in public databases , using CAP3 software (http://seq.cs.iastate.edu/) (Table 1). The resulting “.ace” file was used to study coverage and construct user-friendly alignment views with Mview . To construct the Turbot 3 database, the primitive sequences of Turbot 2 (17,626) were pooled with the 454 contigs (65,472) and then clustered using CAP3 software. The resulting contigs and singletons were annotated using AutoFact (http://www.bch.umontreal.ca/Software/AutoFACT.htm), BLASTN and BLASTX with databases nr, UniProt, UniRef, COG, KEGG, PFam, LSU and SSU. Results were uploaded to a MySQL database and a portal web was created.
To study the different pathways found in the Turbot 3 database the DAVID web tool was used [120, 121]. After the selection of the pathways of interest, a list of reference genes was downloaded from the NCBI RefSeq database and BLASTed against the Turbot 3 database. A gene was considered present in our database if its reference sequence had a match with an e-value cut off ≤ 1,00E-5 and hit length ≥50. To make the colour pathway diagrams (Additional file 2 and Additional file 4) the KEGG mapper tool http://www.genome.jp/kegg/tool/map_pathway2.html was used [122, 123]. Due to the lack of a D. rerio Chemokine signaling pathway in KEGG website the human version was used for Additional file 2. In Additional file 4, the Progesterone-mediated oocyte maturation pathway from D. rerio given by KEGG website is labeled as Xenopus oocyte. This label is kept in the figure.
Microsatellites and SNPs
For SSR and SNP detection, EST sequences were clustered with CAP3 using default parameters and the resulting “.ace” format assembly file was fed into the corresponding programs. The set of unique sequences was searched for microsatellites using the SPUTNIK program (http://espressosoftware.com/sputnik/). The minimum repeat number used for this search was six for dinucleotide and four for tri-, tetra- and pentanucleotide microsatellites. Microsatellite-containing ESTs were identified as candidates for marker development if they presented enough flanking sequences on either side of the repeats for primer design. Whenever possible, we selected three putative primers using the Primer3 software (http://frodo.wi.mit.edu/primer3/).
SNP detection was performed with contigs of at least four sequences using the QualitySNP program (http://www.bioinformatics.nl/tools/snpweb/). This program uses three filters for the identification of reliable SNPs (see  for details). SNPs that pass filters 1 and 2 are called real SNPs and those passing all filters are called true SNPs. The resulting files were processed with our own custom Perl programs to extract relevant information. The obtained true SNPs were imported into a MySQL database (http://www.mysql.com). A user-friendly web access interface was designed so that contig graphs are clickable and the output visually refined with color-coded nucleotide views (http://bio-mview.sourceforge.net/). A graphical interface allowing for SNP database search by alleles, contig depth, and annotation was also established in our on line database. Searchable chromatograms for each of the Sanger sequences making up each contig were also included. It should be emphasized that SNPs detected with the help of bioinformatic pipelines are only putative and they should be technically validated.
To ensure identification of new molecular markers, sequences similar to GenBank deposited sequences were filtered out to avoid identification of already known SSR and SNP sequences, especially the ones previously identified by turbot [18, 19, 48, 51, 94, 124, 125].
A custom 2 x 105 K array was printed with turbot sequences from the Turbot 3 database by Agilent Technologies (US). In order to study the orientation of the non-annotated sequences and their possible gene expression, false annotation of genes and identify possible NATs, oligos were designed in both orientations, forward and reverse. Oligo design was done by using Repeat Masker to eliminate low-complexity regions, and then OligoArray 2.1 software to do the design itself . Cross-hybridization between oligos was checked by BLAST searches against the entire Turbot 3 database and oligos with ≥ 3 putative cross-hybridizations were removed. A total number of 96,292 oligos were printed and almost half of the array contained oligos (47,291) also designed with the opposite orientation. This pilot microarray also included all default positive and negative controls defined by the company (1,325 spots).
The same samples of immune tissues used for library construction and Sanger sequencing and those from the brain-pituitary-gonad axis used for 454 sequencing were used for hybridization (in duplicate) with the pilot microarray. A total of four microarrays were used, two for the reproductive system and two for the immune system. Hybridizations were performed at the Universidad de Santiago de Compostela (USC) Functional Genomics Platform by the Agilent Technology Gene Expression Unit using a 1-colour labeling protocol. This method demonstrated very similar performances to the 2-colour protocol [97, 127]. Briefly, 50 ng of total RNA were labelled using the Low Input Quick Amp Labeling Kit, One-Color (Cy3) (Agilent Technologies, USA). cRNA was prepared for overnight hybridization with the corresponding buffers during 17 h at 65°C and washed on the following day. Hybridized slides were scanned using an Agilent G2565B microarray scanner (Agilent Technologies, USA).
Pilot microarray data processing, filtration, and identification of NATs
The hybridization signal was captured and processed using an Agilent scanner (G2565B, Agilent Technologies, USA). The scanner images were segmented with the Agilent Feature Extraction Software (v9.5) using protocol GE1-v5_95. Extended dynamic range implemented in the Agilent software was applied to avoid saturation in the highest intensity range. Agilent feature extraction produced the raw data for further pre-processing. The processed signal (gProcessed-Signal) value was chosen as statistical for the absolute hybridization signal.
The filtration process was made in two steps. First, the features which did not conform with any of the following well established quality criteria were filtered: (1) non-uniform pixel distributed outliers and population replicate outliers according to the default Agilent feature extraction criteria; (2) features whose ratio between processed signal and their error was below 2; (3) spots not differentiated from background signal (as estimated for each spot); (4) features below the limit where the linear relationship between concentration and intensity was lost according to Spike-In information. The numbers of oligos filtered using this first step is shown in Table 10. Second, two additional filtering criteria were applied: (5) only features with intensity ≥ 100 fluorescence units were kept; (6) features likely to present cross-hybridization were filtered. Table 10 shows the numbers of oligos filtered using the complete filtration process.
For miRNA identification in the Turbot 3 database, a BLASTN search against the miRBase v.18 database (http://www.mirbase.org/) was used. The ten best matches were selected and are depicted in Table 11.
Statistical analyses were carried out with the statistical language R (2.13.1 version). The GOStats Bioconductor package (version 2.18) was used to perform the analysis of GO Terms.
We thank the CCiT-UB staff for their collaboration in sequencing turbot RNA samples. We also thank the Cluster of Aquaculture of Galicia (CETGA) for providing the facilities for the infection trial and doing the sampling and collection of organs for the cDNA library and 454 run. The current work was granted by the Spanish Government thanks to a Consolider Project (Project Aquagenomics, ref. CDS2007-0002) and to projects AGL2006-13158-C03 and AGL2009-13282-C01 and C02. LR was supported by an Aquagenomics postdoctoral contract and BGP was supported by an Isidro Parga Pondal research fellowship from the Xunta de Galicia (Spain). Authors want to thank M.J. Redondo (IATS), María Vázquez (CETGA) and Ana Paula Losada (USC) for their assistance with the infection trial.
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