The objective within the AGENAE French program was to initiate a high-throughput cDNA sequencing program of a 38-tissue normalised library and generate a diverse microarray for transcriptome analysis in pig species.

In pigs, like in other domestic animals, breeding and product quality improvement require the control of several different traits (reproduction, nutrition, health and welfare). It is thus necessary to improve our knowledge of the major physiological functions and their interactions. For this purpose, the French National Institute for Agricultural Research (INRA) [1] has launched a genomic research program, AGENAE (Analyse du GENome des Animaux d'Elevage) [2] for the identification and the functional and genetic characterisation of a large number of genes in cattle, pigs, chicken and trout [3].

With the shift from map-based towards sequence-based gene discovery, the prevailing approach for creating transcription maps has become the generation of Expressed Sequence Tags [4]. In pigs, the first EST project [5] and first large-scale EST project were reported [6] about ten years ago. Subsequently, several research groups have generated ESTs from cDNA libraries constructed from either a single porcine tissue or a limited number of tissues related to a stage of development or a function, such as anterior pituitary [7,8], backfat [9], brain [10], liver [11], skeletal muscle [12-14], immune system tissues [15], reproductive tissues [8,16,17] and embryo [17-19]. The construction of full-length cDNA libraries was reported more recently [20-22].

To date, the construction of several pig arrays have been reported. Some of them, with various supports, contain 1 to 4000 cDNA from specific libraries: brain tissue[10] (GEO database accession number GPL336), muscle [23] (GPL518)[24] (GPL2731), embryo [25] (GPL1209), immune system cells [26,27] (GPL1624), but others aim at a generic analysis (10 to 20000 genes) of pig transcriptome with glass slides of in situ-synthesised oligonucleotides (Affymetrix, GPL3533), spotted oligonucleotides (Operon-Qiagen set) (GPL 1881, GPL3461, GPL3707) or cDNAs (GPL3585, GPL3608).

We report here the construction of a pig multi-tissue cDNA library, its sequencing and analysis, and the generation of a 9K nylon micro-array public tool for large scale expression profiling experiments.

Table 1 Description of the different libraries

PCR amplification with specific primers for the external control genes SRG3, luciferase and I11a (abundant, medium and low-frequency) was used to check the normalisation process (Figure 1). Southern blot experiments demonstrated that the abundance of actin gene and of the abundant spike mRNA SRG3 have been greatly reduced by the normalisation process (data not shown). In addition, quantitative PCR experiments (data not shown) demonstrated that the representation of SRG3 had been reduced 5800 times, the representation of luciferase reduced 4 times and the representation of I11a increased 1.5 times. In the normalised library, the representation of the external controls was estimated as follows: SRG3 = 0.0009%, luciferase = 0.0125%, I11a = 0.0008% as compared to the initial frequencies of 10%, 0.1% and 0.001% respectively.

Figure 1 Control of the normalisation procedure. Normalisation process efficiency was tested by using specific amplification of the control genes SRG3, Luciferase and I11a. Thirty cycles of amplification have been performed, using indicated amounts of plasmid (more ...)

After a first round of sequencing, the library was subtracted with the 8736 already-sequenced clones. The subtracted library contained 60 000 clones. The quality of the subtraction was assessed by the sequencing of 384 clones (see below).

Table 2 Number of sequenced and released ESTs from the two Agenae libraries

Figure 2 Evolution of redundancy rate. Redundancy of the library was calculated as follows: redundance = 1 - (number of genes/number of clones). The number of genes is estimated by the number of contigs obtained at the end of the SIGENAE processing chain.

In the first 384-sequences from the subtracted library, the proportion of 28S RNA sequences had decreased towards 0.52% and 95.1% sequences were new, in comparison with the normalised library. The proportion of empty clones was still about 2%. A total of 14976 clones were then 5'-end sequenced and 12778 (85.3%) sequences were released in the EMBL-EBI nucleotide database (Table 2) [28]. Sequencing was then stopped: the redundancy had reached 39% (Figure 2). The EMBL accession numbers are listed in supplemental data 1. PolyA was detected in 41.5% of the sequences. Polyadenylation signal was detected in 52.9% of the polyA-containing sequences. The sequence of the medium-frequency external control (luciferase) was present 5 times. The other two external control sequences were still not detected.

The library construction method (through the excess of oligo(dT) during the first reverse transcription) led to short polyA 3'-end stretches, allowing almost the same validity rate of the sequences either from 5' (82.2%) or 3' (80.9%)-end. Thus, even if 3'-end sequencing is useful to distinguish genes in a closely related family as the 3'-end non coding regions are more divergent, the 5'-end sequencing strategy was favoured to provide better annotated clones.

Figure 3 Histogram of contig depths. The proportion of Agenae-specific contigs (purple squares) and public porcine contigs (blue squares) is represented in the different contig size classes.

The number of contigs highly depends on the parameters used when assembling the sequences. The TIGR pig clustering[31], with nearly the same amount of data, lead to 64,746 contigs and 88,274 singletons. Careful annotation of the contigs and the next completion of the pig genome sequence may show that paralogous genes are sometimes clustered and that, on the contrary, overlapping contigs may have been split up. UniGene clustering [32] lead to 32,711 contigs and 7,230 singletons. The low number of singletons in UniGene assembly is probably due to the fact UniGene does not use part of the available singletons, as was already noticed with trout data [3].

Table 3 20 deepest contigs

The analysis of the sequences obtained from the normalised library revealed a contamination by 28S ribosomal RNA This type of contamination has already been described in cDNA libraries [35]. This sequence has been over-represented in the subtraction driver and the analysis of the sequences from the subtracted library reveals the presence of 127 out of 14,976 28S ribosomal clones (0.85%). The proportion of this contamination has then been reduced by about 5.

The twenty deepest AGENAE-specific-contigs are listed in Table 4. No hit is found with pig proteins. The best swissprot hit of 16 contigs are with either primates (human, or chimpanzee) rodent (mouse, rat) or other mammals (dog, bovine). Four contigs do not have any swissprot hit. As many tissues were mixed to construct the libraries, without tagging of the cDNAs, specific-tissue sequences were searched. The results are listed in Table 5. The TrainA protein, which is only expressed in epididymis is found [36]. So are GDF9, which is specific to ovary and over-expressed in oocytes [37] and specific mRNA for heart, pituitary gland, muscle or stomach, demonstrating that the multi-tissue library strategy was efficient to get low-redundancy information from a large set of tissues.

Table 4 20 deepest Agenae specific contigs

Table 5 tissue-specific contigs

Figure 4 Hybridisation of the generic 9K pig microarray with an oligonucleotide probe. The array is composed of 64 (16*4) blocks of 144 (12*12) spots. At the four corners of each block, 3 external controls and a negative control (upper right corner) are spotted. (more ...)

This nylon array is a valuable tool for transcriptome analysis. The use of radiolabelled complex probes allows to detect low-expressed mRNAs using small total RNA amounts (about 100 ng of total RNA) [38]. Such arrays have been used in several studies on human cancer [39] trout reproduction [40] and pig transcriptome [24], Rigaldie, E and Liaubet, L personal communications).

Figure 5 Gene Ontology annotation of the generic 9K pig microarray. 5.1, 5.2 and 5.3 indicate the distribution of the annotated contigs into molecular function, biological process and cellular component, respectively.

We constructed a pig multi-tissue cDNA library which has been successfully normalised and subtracted. This library is derived from the most diverse tissue representation to date. It provides a large set of clones, with limited redundancy but good representation of the complex set of initial tissues. The 24,449 sequences allowed a precise characterisation of the library and contributed to international cDNA sequencing effort.

The 9K nylon cDNA microarray is now used in several gene expression profiling projects, in pig health, reproduction and meat quality.

Total RNA was extracted, using the Chomczynski method [41] and controlled (integrity, reverse transcription efficiency) resulting in 38 high quality preparations.

These total RNA were pooled into 6 groups in equal proportions, according to biological functions (Table 1): brain, digestive function, glands, heart and muscle, male reproduction, female reproduction. PolyA+ mRNA was extracted from 300 μg of RNA from these pools.

As a control, 3 exogenous polyA+ mRNAs ("spikes") obtained by in vitro transcription of the corresponding cDNA sequences of SRG3 (A. thaliana, X98376), luciferase (P. pyralis, CVU03687) and I11a (A. thaliana, Y10291) were added to the polyA+ RNAs of each library in different amounts: 6.578 ng, 104.15 pg and 0.274 pg/μg pig RNA respectively. These concentrations correspond to the respective estimated frequencies of 0.5, 50 and 5000 copies of mRNA per cell.

The normalisation was achieved through the reassociation of an excess of cDNA inserts, amplified by PCR, with single-stranded plasmid circles, obtained from the starting library (I) [42]. Single-stranded plasmids were generated in vivo and purified by chromatography on hydroxyapptite (HAP). One ng of the single-stranded library was used in a high-fidelity PCR (Qiagen Taq Polymerase, 250 UI, reference 201203) with T3 and T7 primers primer (see Additional file 2). 500 ng of PCR products were mixed with 50 ng of the single-stranded library and allowed to hybridise for 22 hours (Cot = 5). The remaining single-stranded circles were purified by HAP chromatography, converted into double-stranded plasmids with T7 sequenase (USB, reference 707752), and electroporated into DH10B bacteria. This led to the normalised library (N) The bound double-stranded fraction was recovered from the HAP column and used to generate a mini-library (A) enriched for abundant mRNAs.

The rate of empty clones and the average size of the inserts were estimated by a PCR amplification of the inserts from 96 clones, by using primers (M13/24 and M13Raster, see Additional file 2) flanking the vector-cloning sites primer.

The subtraction was achieved in a similar way using a Cot = 50: 50 ng of the single-stranded normalised library was reassociated with 2.5 μg of PCR products (primers M13/24 and M13Raster, see Additional file 2) generated from the 8736 sequenced clones of the normalised library. In order to eliminate one over-represented contig, 10% of these PCR products were generated from 4 clones chosen to represent the consensus sequence of this contig.

The quality of the normalisation or subtraction was assessed using the external controls in southern blot and PCR experiments. For southern blot experiment, 500 ng of the I, A and N libraries were separated by electrophoresis on a 1% agarose gel and blotted onto a nylon membrane. The blot was hybridised with a labelled probe corresponding to the external controls. PCR experiments were done with specific primers for external controls (see Additional file 2): 30 cycles of amplification were performed, using different amounts of plasmid DNA from each library (A, I or N) as a template. The PCR products were analysed on 1% agarose gel. The sequencing of 96 to 384 randomly picked clones was also used to assess the quality of the normalisation or subtraction processes.

A total of 23712 clones were sequenced, following plasmid DNA preparation, from either 5' or both ends by MilleGen^® Biotechnologies [45] using M13 (-43) or M13R (-47) primers (see Additional file 2) andBigDye V3.1 (Applied Biosystem) or ET terminator (Amersham) chemistries.

The identification and annotation retrieval of the 20 deepest contigs of the assembly and of the 20 deepest AGENAE specific contigs was done by SQL requests on the SIGENAE database. For the deepest contigs, the sequences from Agenae libraries were counted.

Sequences corresponding to putative "tissue specific" proteins in the normalised or subtracted library were identified using a best blast hit strategy for the approximation of ortholog pig genes. The tissue-specificity was documented by literature and the contig containing the nucleotide sequence of the gene referenced in the publication was searched through the SIGENAE WEB interface. If the publication of the sequence was posterior to the SIGENAE assembly, a blastn of the sequence against public_pig_contigV3 database was performed and the contig with a 0 E-value considered. Then an AGENAE sequence was identified in the contig.

The following descending order criteria were examined:

- the size of the insert had to be between 0.7 and 1.5 kb-long

- priority was given to a Agenae clone

- priority was given to the longest-insert Agenae clone

Other clones came from home-made libraries and chosen by INRA researchers.

To assess the quality of the different steps from sequencing to rearraying, the 4 corners of all plates were controlled by sequencing (4 clones/corner); the obtained sequences were compared with the expected sequences.

AB carried out the library construction, normalisation and subtraction. EI performed the sequence processing, assembly and the gene ontology annotation and assisted the array design. KH carried out the clone rearrangement, PCR amplification, array spotting and quality control. FB participated in the clone picking and arraying and gave technical assistance for the robotic management. MBS and MFB welcomed AB in their laboratory and helped her for the construction of the library. FH initiated the study and supervised the experiments. GTK performed the design of the library, the preliminary experiments for library construction, the sequence data analysis, the array design and wrote the manuscript. All authors read and approved the final manuscript.

We thank the colleagues who provided the different pig tissues and/or RNA extracts. We thank Laurent Mazzolini and Véronique Duranthon for providing external controls. We thank Philippe Mulsant for his help during Gwenola Tosser-Klopp's maternity leave, Sandrine Villeger and Cédric Cabau for technical help. We are grateful to Béatrice Loriod and her colleagues from CIML for their help with microarray spotting technique. This work was part of the French National program AGENAE.