De Novo Sequencing Research Articles

De Novo Sequencing, Assembly, and Analysis of the Root Transcriptome of Persea americana (Mill.) in Response to Phytophthora cinnamomi and Flooding

Avocado is a diploid angiosperm containing 24 chromosomes with a genome estimated to be around 920 Mb. It is an important fruit crop worldwide but is susceptible to a root rot caused by the ubiquitous oomycete Phytophthora cinnamomi. Phytophthora root rot (PRR) causes damage to the feeder roots of trees, causing necrosis. This leads to branch-dieback and eventual tree death, resulting in severe losses in production. Control strategies are limited and at present an integrated approach involving the use of phosphite, tolerant rootstocks, and proper nursery management has shown the best results. Disease progression of PRR is accelerated under high soil moisture or flooding conditions. In addition, avocado is highly susceptible to flooding, with even short periods of flooding causing significant losses. Despite the commercial importance of avocado, limited genomic resources are available. Next generation sequencing has provided the means to generate sequence data at a relatively low cost, making this an attractive option for non-model organisms such as avocado. The aims of this study were to generate sequence data for the avocado root transcriptome and identify stress-related genes. Tissue was isolated from avocado infected with P. cinnamomi, avocado exposed to flooding and avocado exposed to a combination of these two stresses. Three separate sequencing runs were performed on the Roche 454 platform and produced approximately 124 Mb of data. This was assembled into 7685 contigs, with 106 448 sequences remaining as singletons. Genes involved in defence pathways such as the salicylic acid and jasmonic acid pathways as well as genes associated with the response to low oxygen caused by flooding, were identified. This is the most comprehensive study of transcripts derived from root tissue of avocado to date and will provide a useful resource for future studies.

De-novo RNA sequencing and metabolite profiling to identify genes involved in anthocyanin biosynthesis in Korean black raspberry (Rubus coreanus Miquel).

The Korean black raspberry (Rubus coreanus Miquel, KB) on ripening is usually consumed as fresh fruit, whereas the unripe KB has been widely used as a source of traditional herbal medicine. Such a stage specific utilization of KB has been assumed due to the changing metabolite profile during fruit ripening process, but so far molecular and biochemical changes during its fruit maturation are poorly understood. To analyze biochemical changes during fruit ripening process at molecular level, firstly, we have sequenced, assembled, and annotated the transcriptome of KB fruits. Over 4.86 Gb of normalized cDNA prepared from fruits was sequenced using Illumina HiSeq™ 2000, and assembled into 43,723 unigenes. Secondly, we have reported that alterations in anthocyanins and proanthocyanidins are the major factors facilitating variations in these stages of fruits. In addition, up-regulation of F3′H1, DFR4 and LDOX1 resulted in the accumulation of cyanidin derivatives during the ripening process of KB, indicating the positive relationship between the expression of anthocyanin biosynthetic genes and the anthocyanin accumulation. Furthermore, the ability of RcMCHI2 (R. coreanus Miquel chalcone flavanone isomerase 2) gene to complement Arabidopsis transparent testa 5 mutant supported the feasibility of our transcriptome library to provide the gene resources for improving plant nutrition and pigmentation. Taken together, these datasets obtained from transcriptome library and metabolic profiling would be helpful to define the gene-metabolite relationships in this non-model plant.

Genome sequencing of four Aureobasidium pullulans varieties: biotechnological potential, stress tolerance, and description of new species.

BackgroundAureobasidium pullulans is a black-yeast-like fungus used for production of the polysaccharide pullulan and the antimycotic aureobasidin A, and as a biocontrol agent in agriculture. It can cause opportunistic human infections, and it inhabits various extreme environments. To promote the understanding of these traits, we performed de-novo genome sequencing of the four varieties of A. pullulans.ResultsThe 25.43-29.62 Mb genomes of these four varieties of A. pullulans encode between 10266 and 11866 predicted proteins. Their genomes encode most of the enzyme families involved in degradation of plant material and many sugar transporters, and they have genes possibly associated with degradation of plastic and aromatic compounds. Proteins believed to be involved in the synthesis of pullulan and siderophores, but not of aureobasidin A, are predicted. Putative stress-tolerance genes include several aquaporins and aquaglyceroporins, large numbers of alkali-metal cation transporters, genes for the synthesis of compatible solutes and melanin, all of the components of the high-osmolarity glycerol pathway, and bacteriorhodopsin-like proteins. All of these genomes contain a homothallic mating-type locus.ConclusionsThe differences between these four varieties of A. pullulans are large enough to justify their redefinition as separate species: A. pullulans, A. melanogenum, A. subglaciale and A. namibiae. The redundancy observed in several gene families can be linked to the nutritional versatility of these species and their particular stress tolerance. The availability of the genome sequences of the four Aureobasidium species should improve their biotechnological exploitation and promote our understanding of their stress-tolerance mechanisms, diverse lifestyles, and pathogenic potential.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-549) contains supplementary material, which is available to authorized users.

Comparative study of five Legume species based on De Novo Sequence Assembly and Annotation

Legume species are an important oilseed crop in tropical and subtropical regions of the world. Recently, next-generation sequencing technology, termed RNA-seq, has provided a powerful approach for analysing the Transcriptome. This study is focus on RNA-seq of five legume species which are Arachis hypogaea L. (The peanut) of SRR1212866, Cicer arietinum L. of SRR627764, Phaseolus vulgaris L. of SRR1283084, Trigonella foenum-graecum L. of SRR066197 and Vicia sativa L. of SRR403901 from NCBI database. Comparative study focuses on various important features like; reads were generated with N50, sequence assembly contigs which is further searched with known proteins and genes; among these, how many genes were annotated with gene ontology (GO) functional categories and sequences mapped to pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). These data will be useful for gene discovery and functional studies and the large number of transcripts reported in the current study will serve as a valuable genetic resource of these five legume species.

Comparative study of five Legume species based on De Novo Sequence Assembly and Annotation

Comparative study of five Legume species based on De Novo Sequence Assembly and Annotation

Filtering of MS/MS data for peptide identification.

BackgroundThe identification of proteins based on analysis of tandem mass spectrometry (MS/MS) data is a valuable tool that is not fully realized because of the difficulty in carrying out automated analysis of large numbers of spectra. MS/MS spectra consist of peaks that represent each peptide fragment, usually b and y ions, with experimentally determined mass to charge ratios. Whether the strategy employed is database matching or De Novo sequencing, a major obstacle is distinguishing signal from noise. Improved ability to distinguish signal peaks of low intensity from background noise increases the likelihood of correctly identifying the peptide, as valuable information is preserved while extraneous information is not left to mislead.ResultsThis paper introduces an automated noise filtering method based on the construction of orthogonal polynomials. By subdividing the spectrum into a variable number (3 to 11) of bins, peaks that are considered "noise" are identified at a local level. Using a De Novo sequencing algorithm that we are developing, this filtering method was applied to a published dataset of more than 3000 mass spectra and an original dataset of more than 300 spectra. The samples were peptides from purified known proteins; therefore, the solutions could be compared to the correct sequences and the peaks corresponding to b, y and other fragments of significance could be identified. The same procedure was applied using two other published filtering methods. The ratios of the number of significant peaks that were preserved relative to the total number of peaks in each spectrum were determined. In the event that filtering out too many or too few signal peaks can lead to inaccuracy in sequence determination, the percentage of amino acid residues in the correct positions relative to the total number of amino acid residues in the correct sequence was also calculated for each sequence determined.ConclusionsThe results show that an orthogonal polynomial-based method of distinguishing signal peaks from background in mass spectra preserves a greater portion of signal peaks than compared methods, improving accuracy in sequence determination.

An immunodominant La/SSB autoantibody proteome derives from public clonotypes

The La/SSB autoantigen is a major target of long-term humoral autoimmunity in primary Sjögren's Syndrome (SS) and systemic lupus erythematosus. A majority of patients with linked anti-Ro60/Ro52/La responses target an NH2-terminal epitope designated LaA that is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. In this study, we used high-resolution Orbitrap mass spectrometry to determine the clonality, isotype and V-region sequences of LaA-specific autoantibodies in seven patients with primary SS. Anti-LaA immunoglobulin (Ig)Gs purified from polyclonal sera by epitope-specific affinity chromatography were analysed by combined database and de-novo mass spectrometric sequencing. Autoantibody responses comprised two heavily mutated IgG1 kappa-restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3-30 heavy chain paired with IGKV3-15 light chain and the second by an IGHV3-43/IGKV3-20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity-determining regions, consistent with a common breach of B cell tolerance followed by antigen-driven clonal selection. The discovery of public clonotypic autoantibodies directed against an immunodominant epitope on La, taken together with recent findings for the linked Ro52 and Ro60 autoantigens, supports a model of systemic autoimmunity in which humoral responses against protein-RNA complexes are mediated by public sets of autoreactive B cell clonotypes.

Genome and transcriptome sequencing of the halophilic fungus Wallemia ichthyophaga: haloadaptations present and absent

BackgroundThe basidomycete Wallemia ichthyophaga from the phylogenetically distinct class Wallemiomycetes is the most halophilic fungus known to date. It requires at least 10% NaCl and thrives in saturated salt solution. To investigate the genomic basis of this exceptional phenotype, we obtained a de-novo genome sequence of the species type-strain and analysed its transcriptomic response to conditions close to the limits of its lower and upper salinity range.ResultsThe unusually compact genome is 9.6 Mb large and contains 1.67% repetitive sequences. Only 4884 predicted protein coding genes cover almost three quarters of the sequence. Of 639 differentially expressed genes, two thirds are more expressed at lower salinity. Phylogenomic analysis based on the largest dataset used to date (whole proteomes) positions Wallemiomycetes as a 250-million-year-old sister group of Agaricomycotina. Contrary to the closely related species Wallemia sebi, W. ichthyophaga appears to have lost the ability for sexual reproduction. Several protein families are significantly expanded or contracted in the genome. Among these, there are the P-type ATPase cation transporters, but not the sodium/ hydrogen exchanger family. Transcription of all but three cation transporters is not salt dependent. The analysis also reveals a significant enrichment in hydrophobins, which are cell-wall proteins with multiple cellular functions. Half of these are differentially expressed, and most contain an unusually large number of acidic amino acids. This discovery is of particular interest due to the numerous applications of hydrophobines from other fungi in industry, pharmaceutics and medicine.ConclusionsW. ichthyophaga is an extremophilic specialist that shows only low levels of adaptability and genetic recombination. This is reflected in the characteristics of its genome and its transcriptomic response to salt. No unusual traits were observed in common salt-tolerance mechanisms, such as transport of inorganic ions or synthesis of compatible solutes. Instead, various data indicate a role of the cell wall of W. ichthyophaga in its response to salt. Availability of the genomic sequence is expected to facilitate further research into this unique species, and shed more light on adaptations that allow it to thrive in conditions lethal to most other eukaryotes.

An aeroplysinin-1 specific nitrile hydratase isolated from the marine sponge Aplysina cavernicola.

A nitrile hydratase (NHase) that specifically accepts the nitrile aeroplysinin-1 (1) as a substrate and converts it into the dienone amide verongiaquinol (7) was isolated, partially purified and characterized from the Mediterranean sponge Aplysina cavernicola; although it is currently not known whether the enzyme is of sponge origin or produced by its symbiotic microorganisms. The formation of aeroplysinin-1 and of the corresponding dienone amide is part of the chemical defence system of A. cavernicola. The latter two compounds that show strong antibiotic activity originate from brominated isoxazoline alkaloids that are thought to protect the sponges from invasion of bacterial pathogens. The sponge was shown to contain at least two NHases as two excised protein bands from a non denaturating Blue Native gel showed nitrile hydratase activity, which was not observed for control samples. The enzymes were shown to be manganese dependent, although cobalt and nickel ions were also able to recover the activity of the nitrile hydratases. The temperature and pH optimum of the studied enzymes were found at 41 °C and pH 7.8. The enzymes showed high substrate specificity towards the physiological substrate aeroplysinin-1 (1) since none of the substrate analogues that were prepared either by partial or by total synthesis were converted in an in vitro assay. Moreover de-novo sequencing by mass spectrometry was employed to obtain information about the primary structure of the studied NHases, which did not reveal any homology to known NHases.

De Novo Sequencing and Transcriptome Analysis of Wolfiporia cocos to Reveal Genes Related to Biosynthesis of Triterpenoids

Wolfiporia cocos Ryvarden et Gilbertson is a saprophytic fungus in the Basidiomycetes. Its dried sclerotium is widely used as a traditional crude drug in East Asia. Especially in China, the dried sclerotium is regarded as the silver of the Chinese traditional drugs, not only for its white color, but also its medicinal value. Furthermore, triterpenoids from W. cocos are the main active compounds with antitumor and anti-inflammatory activity. Biosynthesis of the triterpenoids has rarely been researched. In this study, the de novo sequencing of the mycelia and sclerotia of W. cocos were carried out by Illumina HiSeq 2000. A total of 3,484,996,740 bp from 38,722,186 sequence reads of mycelia, and 3,573,921,960 bp from 39,710,244 high quality sequence reads of sclerotium were obtained. These raw data were assembled into 60,354 contigs and 40,939 singletons, and 56,938 contigs and 37,220 singletons for mycelia and sclerotia, respectively. The transcriptomic data clearly showed that terpenoid biosynthesis was only via the MVA pathwayin W. cocos. The production of total triterpenoids and pachymic acid was examined in the dry mycelia and sclerotia. The content of total triterpenoids was 5.36% and 1.43% in mycelia and sclerotia, respectively, and the content of pachymic acid was 0.458% and 0.174%. Some genes involved in the triterpenoid biosynthetic pathway were chosen to be verified by qRT-PCR. The unigenes encoding diphosphomevalonate decarboxylase (Unigene 20430), farnesyl diphosphate synthase (Unigene 14106 and 21656), hydroxymethylglutaryl-CoA reductase (NADPH) (Unigene 6395_All) and lanosterol synthase (Unigene28001_All) were upregulated in the mycelia stage. It is likely that expression of these genes influences the biosynthesis of triterpenoids in the mycelia stage.

Novel grass pollen antigens are the major inducers of Th2 cytokine producing T cells in allergic individuals (P6039)

Abstract T cells play an important role in the pathogenesis of allergic diseases. However, potential immunogens of allergenic T cell responses have traditionally been limited to those that induce IgE responses. Timothy grass (TG) pollen is an inhaled allergen for which major IgE-reactive allergens have also been shown to trigger Th2 responses. Here we examined whether other TG pollen proteins are recognized by Th2 responses independently of IgE-reactivity. A TG pollen extract was analyzed by 2D-gel electrophoresis and IgE/IgG immunoblots using pooled sera from allergic donors. Mass spectrometry in combination with de-novo sequencing of the whole TG pollen transcriptome identified 93 novel proteins for further study, 64 of which were not targeted by IgE. Peptides from the novel TG proteins were screened for T cell reactivity in PBMCs from allergic donors. Strong IL-5 production was detected in response to peptides from several novel proteins, most of which were not targeted by IgE. Responses against the dominant novel epitopes were associated with memory T cells and could be detected directly ex vivo after Th2 cell enrichment. These findings demonstrate that a combined unbiased transcriptomic-, proteomic-, and immunomic approach identifies a greatly broadened repertoire of protein antigens targeted by T cells involved in allergy pathogenesis. The discovery of proteins that induce Th2 cells but are not IgE reactive may allow the development of safer immunotherapeutic strategies.

High-Confidence de Novo Peptide Sequencing Using Positive Charge Derivatization and Tandem MS Spectra Merging

De novo peptide sequencing holds great promise in discovering new protein sequences and modifications but has often been hindered by low success rate of mass spectra interpretation, mainly due to the diversity of fragment ion types and insufficient information for each ion series. Here, we describe a novel methodology that combines highly efficient on-tip charge derivatization and tandem MS spectra merging, which greatly boosts the performance of interpretation. TMPP-Ac-OSu (succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide) was used to derivatize peptides at N-termini on tips to reduce mass spectra complexity. Then, a novel approach of spectra merging was adopted to combine the benefits of collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation. We applied this methodology to rat C6 glioma cells and the Cyprinus carpio and searched the resulting peptide sequences against the protein database. Then, we achieved thousands of high-confidence peptide sequences, a level that conventional de novo sequencing methods could not reach. Next, we identified dozens of novel peptide sequences by homology searching of sequences that were fully backbone covered but unmatched during the database search. Furthermore, we randomly chose 34 sequences discovered in rat C6 cells and verified them. Finally, we conclude that this novel methodology that combines on-tip positive charge derivatization and tandem MS spectra merging will greatly facilitate the discovery of novel proteins and the proteome analysis of nonmodel organisms.

Correction: Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Correction: Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Identification and Characterization of a Tetramethylpyrazine Catabolic Pathway in Rhodococcus jostii TMP1

At present, there are no published data on catabolic pathways of N-heterocyclic compounds, in which all carbon atoms carry a substituent. We identified the genetic locus and characterized key reactions in the aerobic degradation of tetramethylpyrazine in Rhodococcus jostii strain TMP1. By comparing protein expression profiles, we identified a tetramethylpyrazine-inducible protein of 40 kDa and determined its identity by tandem mass spectrometry (MS-MS) de novo sequencing. Searches against an R. jostii TMP1 genome database allowed the identification of the tetramethylpyrazine-inducible protein-coding gene. The tetramethylpyrazine-inducible gene was located within a 13-kb genome cluster, denominated the tetramethylpyrazine degradation (tpd) locus, that encoded eight proteins involved in tetramethylpyrazine catabolism. The genes from this cluster were cloned and transferred into tetramethylpyrazine-nondegrading Rhodococcus erythropolis strain SQ1. This allowed us to verify the function of the tpd locus, to isolate intermediate metabolites, and to reconstruct the catabolic pathway of tetramethylpyrazine. We report that the degradation of tetramethylpyrazine is a multistep process that includes initial oxidative aromatic-ring cleavage by tetramethylpyrazine oxygenase, TpdAB; subsequent hydrolysis by (Z)-N,N'-(but-2-ene-2,3-diyl)diacetamide hydrolase, TpdC; and further intermediate metabolite reduction by aminoalcohol dehydrogenase, TpdE. Thus, the genes responsible for bacterial degradation of pyrazines have been identified, and intermediate metabolites of tetramethylpyrazine degradation have been isolated for the first time.

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.

De Novo Sequencing and Transcriptome Analysis of the Central Nervous System of Mollusc Lymnaea stagnalis by Deep RNA Sequencing

The pond snail Lymnaea stagnalis is among several mollusc species that have been well investigated due to the simplicity of their nervous systems and large identifiable neurons. Nonetheless, despite the continued attention given to the physiological characteristics of its nervous system, the genetic information of the Lymnaea central nervous system (CNS) has not yet been fully explored. The absence of genetic information is a large disadvantage for transcriptome sequencing because it makes transcriptome assembly difficult. We here performed transcriptome sequencing for Lymnaea CNS using an Illumina Genome Analyzer IIx platform and obtained 81.9 M of 100 base pair (bp) single end reads. For de novo assembly, five programs were used: ABySS, Velvet, OASES, Trinity and Rnnotator. Based on a comparison of the assemblies, we chose the Rnnotator dataset for the following blast searches and gene ontology analyses. The present dataset, 116,355 contigs of Lymnaea transcriptome shotgun assembly (TSA), contained longer sequences and was much larger compared to the previously reported Lymnaea expression sequence tag (EST) established by classical Sanger sequencing. The TSA sequences were subjected to blast analyses against several protein databases and Aplysia EST data. The results demonstrated that about 20,000 sequences had significant similarity to the reported sequences using a cutoff value of 1e-6, and showed the lack of molluscan sequences in the public databases. The richness of the present TSA data allowed us to identify a large number of new transcripts in Lymnaea and molluscan species.

Constrained De Novo Sequencing of Conotoxins

De novo peptide sequencing by mass spectrometry (MS) can determine the amino acid sequence of an unknown peptide without reference to a protein database. MS-based de novo sequencing assumes special importance in focused studies of families of biologically active peptides and proteins, such as hormones, toxins, and antibodies, for which amino acid sequences may be difficult to obtain through genomic methods. These protein families often exhibit sequence homology or characteristic amino acid content; yet, current de novo sequencing approaches do not take advantage of this prior knowledge and, hence, search an unnecessarily large space of possible sequences. Here, we describe an algorithm for de novo sequencing that incorporates sequence constraints into the core graph algorithm and thereby reduces the search space by many orders of magnitude. We demonstrate our algorithm in a study of cysteine-rich toxins from two cone snail species (Conus textile and Conus stercusmuscarum) and report 13 de novo and about 60 total toxins.

De Novo sequencing and characterization of developing seed transcriptome in two buckwheat species and metabolome profiling

De Novo sequencing and characterization of developing seed transcriptome in two buckwheat species and metabolome profiling

De-Novo Transcriptome Sequencing of a Normalized cDNA Pool from Influenza Infected Ferrets

The ferret is commonly used as a model for studies of infectious diseases. The genomic sequence of this animal model is not yet characterized, and only a limited number of fully annotated cDNAs are currently available in GenBank. The majority of genes involved in innate or adaptive immune response are still lacking, restricting molecular genetic analysis of host response in the ferret model. To enable de novo identification of transcriptionally active ferret genes in response to infection, we performed de-novo transcriptome sequencing of animals infected with H1N1 A/California/07/2009. We also included splenocytes induced with bacterial lipopolysaccharide to allow for identification of transcripts specifically induced by Gram-negative bacteria. We pooled and normalized the cDNA library in order to delimit the risk of sequencing only highly expressed genes. While normalization of the cDNA library removes the possibility of assessing expression changes between individual animals, it has been shown to increase identification of low abundant transcripts. In this study, we identified more than 19000 partial ferret transcripts, including more than 1000 gene orthologs known to be involved in the innate and the adaptive immune response.