BACKGROUND
the co-evolution of host response and pathogen evasion mechanisms  <cit>  drives variation in the response to infectious diseases at individual, population and species levels, as well as at higher order taxonomic units.
immunity-related genes are “tailored to the niche that a species occupies” and exhibit many features of positive selection, including polygeny and clustering of loci, high rates of non-synonymous substitutions , allele and gene conversion, generation of repertoires, rapid evolution, co-evolution, association with diseases and networking  <cit> .
the host response to a pathogen requires concerted action of a huge set of immunity-related genes recently referred to as the immunome  <cit> .
the immunome was first defined as the totality of rearranged antibody and antigen receptor genes present in all living individuals of a species, including variations in the somatic rearrangements  <cit> .
that definition was further adapted to describe the whole set of genes related to both innate and acquired immunity as identified from whole genome sequencing and functional genomics studies  <cit> .
the progress in genome sequencing of human, model and non-model animal species, including livestock species , now permits comparative analysis across many species  <cit> .
such studies have highlighted the divergence in innate immune responses between humans and mice, the most widely-studied experimental animal model  <cit> .
the domestic pig , in the suidae family within the cetartiodactyl order of eutherian mammals, has been used a model in medical research due to its similarity to humans in size and physiology, including comparable digestive, respiratory, and immune systems .
the cetartiodactyl order first appeared 60- <dig> million years ago, while the divergence for rodents and primates  dates from  <dig> and  <dig> million years ago, respectively  <cit> .
primarily as a consequence of the fast sequence evolutionary rate of mouse, the pig has remained considerably more similar at the dna sequence level to humans  <cit> .
however, some immunological features are exceptional in the pig compared to humans and mice.
pigs have an inverted lymph node structure and an unusual route for lymphocyte circulation  <cit> .
there are relatively high numbers of extra-thymic cd4/cd <dig> double positive t-cells  <cit>  and resting t-cells expressing sla class ii molecules  <cit> .
pigs can have high numbers of natural killer cells  <cit>  and γδ cells  <cit> , harbor an unusual diversity of b-cell and antibody repertoire development  <cit> , and have highly heritable variation in immune cell parameters  <cit> .
in pigs as in many other species, the numerous in vivo and in vitro studies on host-pathogen interactions  <cit>  and immunity stimulation  <cit> , are now often based on functional genomics approaches such as transcriptomic approaches  <cit> .
with such rapid accumulation of high-dimensional data on immune response, network models are becoming increasingly important in the interpretation of such experimental data  <cit> .
correlation networks based on immune response data not only permit the identification of common regulatory mechanisms through integration with promoter/flanking sequences, but also provide evidence that un-annotated genes are involved in immune response pathways  <cit> .
thus an important aspect of gene annotation is the integration of structural analysis of rnas and genomes with functional data on transcriptional response to pathogens and immune stimuli.
the purpose of the immune response annotation group  was to explore the porcine immunome by exploiting the recently available genome sequence assembly  <cit> .
a gene list for detailed manual gene annotation using otterlace  <cit>  was compiled using gene ontology  annotation  <cit>  and literature sources.
analyses combined structural, evolutionary and functional approaches.
we report a refined gene structure annotation on greater than  <dig>  genes involved in immunity; data on positive selection pressure of a subset of the proteins predicted to be encoded by these genes; and a correlation network analysis of transcriptomic data from various disease and immunological models.
these three levels of data contribute to a better characterization of the pig immunome and provide a comparative genomic appraisal across mammals.
RESULTS
extensive manual annotation of the genomic complement of porcine immune response genes
the immune response annotation group  members used otterlace  <cit>  to manually annotate over  <dig>  loci in porcine build  <dig> selected based on their membership in immune response processes or gene ontology immune response annotation.
the go term used as an inclusion criterion was “immune system process”; go: <dig>  these structural annotations were transferred over to build  <dig>  and discrepancies were addressed to determine the final annotation results .
members confirmed automated annotation of  <dig> known genes through manual annotation of  <dig>  transcripts and  <dig>  gene models;  <dig>  annotated transcripts contained the full-length protein coding sequence.
twenty-six pseudogenes were also identified during the annotation.
*number of gene objects created in the otterlace annotation system.
** processed and non-processed pseudogenes are included.
# no porcine est or cdna sequence was available to create these transcript predictions.
importantly, the cross-species alignment tools in otterlace allowed annotation of  <dig>  transcripts in the pig genome using only mrna sequence from other species .
such transcripts without specific porcine sequence support were often made using human cdnas , as there are many more human than pig sequences in the databases.
the conservation between human and pig in terms of synteny is three times greater than between human and mouse  <cit> , and the pig is more closely-related at the dna sequence level to humans than either is to the mouse  <cit> .
specifically for immune gene families, recent analysis at the cdna sequence level of pig, mouse and human has shown that the great majority of human genes that were lost through evolution in the mouse were retained in the pig.
conversely, very few mouse genes that were lost through evolution in the human were found in the pig.
comparison of expansion or contraction of orthologous gene families indicated far more similar rates and classes of genes in humans and pigs than in mice.
the conservation of homology and structural motifs of  <dig>  unambiguous orthologs from pigs, mice and humans revealed that the overall mean similarity to human proteins was significantly higher for pigs compared to mouse  <cit> .
the following sections provide summaries of important groups of genes for which the manual annotation revealed new insight.
one important region highly relevant to immune response, the swine leukocyte antigen complex , has been previously annotated in detail  <cit>  and will not be discussed here.
t cell receptor
genes in the tcr complex possess highly repetitive sequences, so that it is difficult to generate correctly reconstructed loci from shotgun sequencing with low redundancy or short-read next-generation sequencing.
therefore, intensive sequencing efforts were applied to the tra/trd  and trb  loci.
the pig trd locus is embedded in tra, and d   and j  segments , and genes encoding the c  region of tcr δ  are located between the v  segments of tcr α/δ  and j genes of tcr α , as observed in other mammals.
all of the human  <dig> jα segments correspond to those of pig, and most of mouse jα can be allocated to orthologs in pig.
these indicate functional similarity of the tcr α molecule between human, mouse and pig  <cit> .
on the other hand, the pig tcr δ gene  has a more complicated structure than those of human and mouse.
pig has at least  <dig> dδ genes, while human and mouse have  <dig> and  <dig>  respectively.
the pig dδ genes are frequently used in functional tcr δ transcripts with up to  <dig> concatenated domains  <cit> .
thus, the pig can generate a high diversity of tcr δ chain molecules to cope with antigens, which may be related to the fact that the percentage of γδ t cells in peripheral blood is much higher in pig than in human and mouse  <cit> .
as for trb, pig has  <dig> functional dβ-jβ-cβ units, while human and mouse each have  <dig> units  <cit> .
immunoglobulin
ighv gene diversity is highly restricted, as in cattle, but all known porcine ighv genes belong to a single family, ighv <dig>  whereas all cattle ighv belong to ighv <dig> <cit> .
the lambda light chain  locus on ssc <dig> contains  <dig> iglv gene segments, with  <dig> appearing functional.
the locus is organized into two distinct clusters, a constant -proximal cluster containing iglv <dig> family members, and a c-distal cluster containing iglv <dig> and iglv <dig> family members  <cit> .
the porcine iglv <dig> subgroup genes have recently expanded, suggesting a particularly effective role in immunity to porcine-specific pathogens, especially since iglv expression is nearly exclusively restricted to the iglv <dig> and iglv <dig> <cit> .
the c-distal iglv cluster also contains three non-functional iglv <dig> family members that are orthologous to iglv that are exclusively expressed in cattle  <cit> .
the igl locus contains three tandem iglj–iglc cassettes, two of which are functional, and a fourth iglj with no corresponding iglc.
the kappa light chain  locus on ssc <dig> is comprised of at least  <dig> igkv genes, of which  <dig> are functional and belong to either the igkv <dig> or igkv <dig> gene families, five igkj genes that lie  <dig>  kb downstream, and a single igkc gene  <cit> .
polymorphisms within the individual duroc sow that was genome sequenced revealed alleles that differed by as much as  <dig> percent among igkv genes and as much as eight percent in amino acid sequence among iglv genes.
the porcine immunoglobulin genes have evolved such that specific gene families have expanded and contracted with respect to other species, notably cattle.
the high level of allelic variation found within the antibody light chain loci substantially expands the population diversity of the porcine antibody repertoire  <cit> .
in the kappa locus, in particular, many igkv <dig> family members share specific parts of coding regions, such as complementary determining region  <dig>  between genes but not between alleles.
thus, germline gene conversion may provide a mechanistic basis for the high level of igkv allelic variation.
killer immunoglobulin-like receptor
pigs appear to have a single kir gene in contrast to cattle, horses and primates in which there is an expanded kir gene family  <cit> .
in rodents, the functionally equivalent receptors are encoded by the expanded gene family of killer cell lectin-like receptor  genes of which klra <dig>  has  <dig> paralogues whilst pigs have a single ly <dig> gene  with two putative orthologs.
the limited nk cell repertoire in pigs is not linked with any deficiency in nk cell numbers  <cit>  but there is evidence for a connection between high variability between individual animals and performance under low health status conditions  <cit> .
one would anticipate, given the function of nk cell receptors in recognition of mhc class  <dig> proteins, that there must be some unique feature to this interaction in the pig to allow nk cells to function irrespective of unlinked polymorphism and the sla loci.
immune gene family expansion
a preliminary analysis of immune gene families has previously compared humans, mice and pigs  <cit> .
in the current analysis, artiodactyl-associated families were also included based upon expansions noted in the bovine genome  <cit> .
our porcine genome analyses show that some of these expansions are also present in the porcine genome, indicating an artiodactyl-specific expansion.
other expansions are not present in the pig genome, providing additional support for a ruminant-specific expansion  <cit> .
results are summarized in table  <dig> and full details shown in additional file 1: table s <dig>
* numbers of confirmed pseudogenes are shown in parentheses.
agp and dcr: asialo- glycoprotein and dc receptor; mc: mast cell; sp: serine protease; up: ubiquitin-protein.
the cathelicidin gene family was expanded, with  <dig> genes compared to only one in human and mouse.
the expansion appears to be artiodactyl-specific since cattle also have  <dig> genes  <cit> .
thirty-four beta defensin genes were detected in the swine genome assembly, similar to the human genome , but substantially less than the > <dig> beta-defensin genes reported from cattle.
a recent report annotated  <dig> porcine defensins in the high-throughput genome sequences  pre-assembly , which indicates that our annotation of  <dig> for the current genome assembly adds to the previous annotation of this family.
for this work, we tested the procedure based on hidden-markov model  that was used in the bovine genome project  <cit> , but primarily used our curation protocol established for genome-wide annotation of beta-defensin families in the human, chimpanzee, mouse, rat, and dog  <cit> .
as a more sensitive procedure, hmm could overestimate the functional gene numbers without manual curation.
thus far, we suggest that the composition of the porcine beta-defensin family is more similar to human  than bovine .
a similar result is observed for the c-type lysozyme family in pigs, which has  <dig> genes, while  <dig>   <dig> and  <dig> are found in the bovine, human and mouse genomes, respectively.
thus, our analysis of the second artiodactyl genome indicates that beta-defensin and c-type lysozyme family expansions observed in cattle may be ruminant-specific adaptations  <cit> .
because pigs are omnivores and cows are herbivores, it is tempting to speculate that these differences may be due to different exposure to gut microbiota.
pigs have  <dig> type i interferon  genes, twice the number in human, as well as  <dig> pseudogenes.
cattle have  <dig> type i ifn genes .
this expansion is focused on interferon subtypes ifnw and ifnd; pigs  and cattle  share novel subtypes of ifnd , ifnt , ifnaw  and many more isoforms of ifnw.
thus, expansion of interferon genes is not ruminant-specific as previously proposed  <cit> , although duplication within some specific sub-families appears to be either bovine- or pig-specific.
data presented in table  <dig> represents an expanded analysis of the gene families that were presented in the recent porcine genome paper  <cit> .
four additional gene families, slam superfamily, granzyme/mast cell tryptase/serine protease superfamily, cytidine deaminase-like superfamily and immunity related guanosine triphosphatase superfamily are included.
these new analyses reveal a slight expansion in the slam superfamily in cow and pig relative to human, a relatively large expansion in the granzyme/mast cell tryptase/serine protease superfamily in mouse and cow relative to humans and pigs, and an extremely large expansion in the immunity related guanosine triphosphatase superfamily in mouse relative to the other  <dig> species.
in contrast, the number of cytidine deaminase-like superfamily members is human is twice that found in pigs, mice and cows.
the total number of pig, mouse and cow gene families that have undergone expansion of >25% of family members relative to human are  <dig>   <dig> and  <dig>  respectively .
thus familial gene expansion in pigs relative to humans has occurred at half the rate of mice and cows.
conversely, the total number of gene families in pig, mouse and cow that have undergone contraction of >25% of family members relative to human are  <dig>   <dig> and  <dig>  respectively.
familial gene contraction in pigs relative to humans has occurred at roughly the same rate of mice and cows.
an additional analysis of orthology preservation of familial gene expansion for the four species deriving from the family member expansion analysis revealed that 1: <dig> orthology conservation was found for  <dig> of the  <dig> genes  .
mice had the largest number of unique genes , more than twice the number found in cattle , and more than all of the others combined.
in contrast, pigs have 11-, 5- and 2-fold fewer unique genes compared to the mouse, cow and human, respectively.
pair-wise analysis indicates that pigs and cows share  <dig> genes that are not found in humans or mice .
these genes are members of the bpi superfamily, bpifb <dig> and bpifb9; cathelicidin superfamily cathl <dig> , cathl <dig> , cathl <dig> , cathl <dig> , cathl <dig> , cathl <dig> ; ccl chemokine superfamily, ccl3l2; cd163/wc <dig> superfamily, loc100337197; clect superfamily, klrj <dig> and prg3l1; granzyme/mast cell tryptase/serine protease superfamily, gzmal , mcpt3; immunity related guanosine triphosphatase superfamily, irgcl1; nlr and pyrin superfamily, nlrp12l; and rnase a family, brb and loc <dig> .
furthermore, analysis of three species at a time indicates that humans and pigs share  <dig> genes that are not found in mice; mice and humans share  <dig> genes that are not found in pigs; and mice and pigs share  <dig> genes that are not found in humans .
these conclusions must be tempered with the observation that the porcine genome is still incomplete and additional family members may be discovered.
indeed, in the course of looking for known genes in the current porcine genome build, we identified genes that do not appear in the build  <dig>  assembly; please see additional file 2: table s <dig>  however, these similarities, especially for pig and human, further reinforce the use of the pig as a large animal model of the immune response of humans.
gene duplication
in the course of annotating the immune response gene families shown in table  <dig>  we found indication of gene duplication and pseudogenes in the build  <dig>  assembly.
a summary of putative true gene duplications is shown in additional file 3: table s <dig>  using extreme sequence similarity  as a metric, many duplications  appear to be due to assembly artifacts .
these artifactually duplicated genes fall into  <dig> different categories: on the same chromosome and mapped to the same assembly scaffold, on the same chromosome and mapped to a different assembly scaffold, and on different chromosomes.
the  <dig> duplications that appear on different chromosomes are especially problematic because of the need to assure bac specificity.
one gene, tnfrsf10a, which is listed provisionally here as artifactually duplicated, proved especially problematic since there is equal evidence for both artifactual and true duplication; directed studies will be necessary to determine the nature of this duplication.
evidence for the true duplication of  <dig> immune-related genes: atf <dig>  cd <dig>  cd <dig>  cd <dig>  crp, ddx3x, gstp <dig>  gzma, ifit <dig>  il1b, irgc, itln <dig>  and oas <dig>  and  <dig> non-immune genes, appears unequivocal .
interestingly, the il1b gene duplication, in which evidence for a partial duplication had been reported  <cit> , is unique in mammals.
predicted proteins expressed from the il1b gene and its duplicate  are both  <dig> aa in length, but only 86% identical.
further, their mrnas have different expression patterns in adult and embryonic tissues, and different responses to endotoxin in macrophages .
unigene expression profiling of other true duplicated genes indicates that they are differentially expressed.
for example the macrophage scavenger receptor cd <dig> is expressed primarily in adipose tissue, blood, lung, mammary gland, and ovary, whereas the porcine-restricted duplicated gene with an unknown function, cd68l, has a wider tissue distribution with expression in adipose tissue, adrenal gland, blood, cartilage, heart, intestine, lung, lymph node, muscle, ovary, placenta, skin, spleen, thymus and trachea.
the pattern recognition receptor cd <dig> is highly expressed primarily in adipose tissue, heart, mammary gland and muscle.
the truncated protein cd36l, with an unknown function, is also duplicated in the cow and is expressed at lower levels in blood, bone marrow liver, lung and mammary gland.
the differential expression patterns of these genes support their phylogenetic and functional divergence.
functional annotation of immune genes: finding genes with immune response patterns similar to known immune system genes
co-expression of genes can be used to provide evidence for membership in specific processes, such as immune response, when a substantial proportion of the members of an expression cluster have similar functions.
we collected all data reported for the 24k affymetrix genechip for experiments with an immune component such as infection of tissues or cells with bacteria, viruses, or stimulation with lipopolysaccharide.
using this targeted set of  <dig> chips , which included public data as well as several un-published datasets from our groups, we calculated a within-group correlation for each experimental dataset.
this approach emphasizes the response to pathogen/stimulus .
these correlations were then used in the co-expression network tool biolayout express3d  to generate and visualize a transcriptome network for porcine generic immune response .
using this correlation network, we then identified clusters with go term annotation enrichment related to immune response.
significance of enrichment was determined as described in methods.
in additional file 5: table s <dig>  and additional file 6: table s <dig> , we document these results.
cluster  <dig>  with  <dig> probesets representing at least  <dig> transcripts, was significantly enriched for many go terms relevant to immune response pathways, including type i interferon-mediated signaling pathway and cytokine-mediated signaling pathway, as well as response to virus, and proteasome core complex.
cluster  <dig> is highlighted in figure  <dig>  and includes many immune-related genes such as il <dig>  jak <dig>  irf <dig>  irf <dig>  irf <dig>  ifit  <dig>  ifit <dig>  ifit <dig>  cd <dig>  cd <dig>  cd <dig>  many stat, psmb and casp gene family members, mx <dig>  mx <dig>  cxcl <dig>  ccrl <dig>  wars, slc11a <dig>  and complement genes c1r, c1s, and c <dig>  of these  <dig> probesets,  <dig> are annotated with the go term originally used as a major criterion for the irag gene list , which is a  <dig> -fold enrichment .
this cluster is also three-fold enriched  for porcine orthologs of common immune response genes identified by jenner and young based on a meta-analysis of microarray data from a number of pathogens/ immune stimulations of several human cell types  <cit> .
thus cluster  <dig> clearly is enriched for a large number of genes likely to be involved in the immune response of swine.
importantly, most of these probesets are not already annotated as immune response genes, as only 16% have the go:  <dig> term annotation.
further, the average rna level for these  <dig> probesets across the immune response datasets is shown in figure  <dig>  the pattern is clearly one of activation upon infection or treatment, as global increases in expression levels are seen in many datasets upon immune stimulation.
thus this correlation analysis provides evidence for the involvement of many new genes in the porcine immune response.
a second cluster with enrichment of go: <dig> is cluster  <dig> .
as with cluster  <dig>  cluster  <dig> is also enriched for jenner-young  ir genes as well as genes manually annotated as “innate immune” genes in innatedb .
for the global go annotation, only anti-apoptosis was found significantly enriched .
genes in this smaller cluster of  <dig> members include several negative regulators of innate/inflammatory pathways  .
the pattern of expression of these genes is similar to that of cluster  <dig>  with cluster  <dig> genes clearly activated in lps-stimulated macrophages and in lymph nodes or blood from pigs infected with salmonella .
interestingly, in contrast to cluster  <dig>  several datasets did not show activation of genes represented by the probesets in cluster  <dig> genes on average, including lymph nodes infected with pcv <dig> or prrsv.
a third cluster contains  <dig> probesets , containing probesets representing several t-cell related genes  as well as other immunity genes such as il7r, gzmb and stat <dig>  this cluster appears to document gene expression specific to t cells and neutrophils, as expression is detected in lymph node and whole blood datasets, and absent in macrophages .
the global annotation identified regulation of immune response as well as several t cell receptor terms as enriched in this cluster .
the pattern of expression is not clearly correlated with response to immune stimulation, which is consistent with the observation that these genes are not enriched for j-y ir or innatedb genes, but are enriched for genes with go: <dig> annotation .
finally, cluster  <dig> is enriched only for probesets annotated with the go: <dig> term, and shows early and robust induction in macrophages infected with prrsv and in lymph nodes in animals infected with salmonella enterica serovar typhimurium, but variable low to modest induction in macrophages by lps  and other challenges.
this cluster of  <dig> probesets includes a number representing mhc genes  as well as cfp, cyba, ly <dig> and irf <dig>  comprehensive go annotation shows this cluster is enriched for interferon gamma-mediated signaling pathway, t cell receptor signaling pathway, cytokine-mediated signaling pathway, and immune response genes .
the established gene lists for generic immune responses at the level of the transcriptome can be used to improve the annotation of a large number of genes/transcripts in the porcine genome related to immune response.
especially for cluster  <dig>  which is significantly enriched for probesets annotated as human immune response genes, these data provide foundational information that can be used for human-pig comparisons at several levels.
for example, global comparisons of promoter sequence elements between pig and other species can be performed using draft genome assembly information, as shown recently for an analysis of the cyp27b <dig> gene promoter  <cit> .
previously, substitution of human orthologous promoters was useful in prediction of sets of nfκb target genes in the pig  <cit> .
given the recent expansion of transcriptomic datasets on immune response, especially for those analyzing response to infection of specific tissues or cell types, the pig genome will now be invaluable in bioinformatic approaches to recognize known and novel regulatory motifs in immune response genes.
prior annotation as immune response genes, as demonstrated herein, will provide further confidence for genes clustered by their transcriptomic response to an immune stimulation.
an accelerated rate of evolution for immunity-related genes
as recently reported  <cit> , an analysis of predicted rates of evolutionary change was carried out on a randomly selected subset of  <dig> immunity-related pig proteins from the irag annotated gene set.
this analysis showed rates of positive selection between  <dig>  and  <dig> %, depending on the analysis method .
to confirm and extend this significant increase in the rate of positive selection for immunity-related genes in swine, we analyzed the proteins present in the cluster  <dig>  found in the above analysis to be significantly enriched for probesets annotated as immunity-related genes.
a set of  <dig> proteins was analyzed with the phyleasprog web server  <cit> , and a subset of  <dig> proteins having at least  <dig> orthologs and being compatible with threshold of statistical significance was included in final analyses.
among these  <dig> proteins,  <dig> proteins are under positive selection with q < <dig> , and  <dig> with q < <dig> ; i.e.
15% and 17% respectively.
thus, cluster  <dig> is as rich in positively selected proteins, as was the subset of  <dig> immunity-related pig proteins recently published in the swine genome sequence paper  <cit> .
at the whole genome level, it has been reported that the rate of positive selection, computed on different types of data and different methods, is  <dig> % in human,  <dig> % in chimpanzee  <cit> , close to 5% in cattle, dog and horse , and  <dig> % in pig   <cit> .
our results show a significant increase in the rate of positive selection for immunity-related genes in swine.
these results confirm that positively selected genes in swine are enriched for roles in defense and immunity in mammals, as already shown in human  <cit> , cow  <cit> , five other mammals  <cit> , as well as birds like the zebra finch  <cit> .
other functions are also reported as privileged targets for an accelerated evolutionary rate of related genes in mammals, such as reproduction, taste perception, chemosensory reception  <cit> , and olfaction, as recently shown in pig  <cit> .
by branch-site analysis, we detected an accelerated evolution of several amino acids specific to pig  in  <dig> proteins , including sppl2a, jak <dig>  ppp2r5c, chd- <dig>  tspan <dig>  nmt <dig>  gbp <dig>  hexb, fam26f, lman2l, ankmy <dig>  phf20l <dig>  ddx <dig>  pde8a, lcp <dig>  usp <dig>  slc24a <dig> .
the projections of amino acids under positive selection onto 3d structure of the four proteins casp <dig>  hexb, gbp <dig> and ppp2r5c are shown via phyleasprog web server .
the ppp25rc protein is known as the protein phosphatase  <dig>  regulatory subunit b’, gamma and is  <dig> amino acids long .
within a segment of  <dig> amino acids from position  <dig> to  <dig>  a total of  <dig> amino acids were found under positive selection.
conversely, amino acids were found under purifying selection from position  <dig> to  <dig>  .
the interproscan revealed matches with the protein phosphatase 2a, regulatory b subunit, b <dig> , and an armadillo-type fold from position  <dig> to  <dig> by the superfamily method.
these striking results suggest two potentially distinct subdomains for the protein ppp25rc in swine.
the hexb protein, hexosaminidase b , is  <dig> amino acids long in swine .
hexosaminidase b is the beta subunit of the lysosomal enzyme beta-hexosaminidase that, together with the cofactor gm <dig> activator protein, catalyzes the degradation of the ganglioside gm <dig>  and other molecules containing terminal n-acetylhexosamines.
two amino acids were found under positive selection at positions  <dig> and  <dig>  both amino acids map to the glycoside hydrolase catalytic domain .
at position  <dig>  the amino acid maps also to a beta-hexosaminidase subunit related to beta-n-acetylhexosaminidase activity .
the casp <dig> protein, known as the caspase  <dig>  apoptosis-related cysteine peptidase comprises  <dig> amino acids in swine  and has also been found under accelerated evolution rate in human .
two amino acids were found under positive selection at positions  <dig> and  <dig>  the amino acid at position  <dig> specifically maps to a predicted domain referred to as domain peptidase c <dig>  caspase precursor p <dig>  core-il1bcenzyme .
the gbp <dig> protein is known as the guanylate binding protein  <dig>  interferon-inducible and is  <dig> amino acids long .
a unique amino acid has been found under positive selection at position  <dig>  in the guanylate binding protein, c-terminal domain .
the availability of whole genome sequences has paved the way for renewed approaches to study the molecular signatures of natural selection at unprecedented scales  <cit> .
in addition, amino acids found under positive selection are highly interesting candidates to target for further biological analyses and understanding of the link between structures and functions.
in genetic studies aimed at identifying nucleotide polymorphisms involved in the variation of target traits, the analysis of evolutionary constraints at candidate mutations should provide a fundamental, additional layer of information.
CONCLUSIONS
our computationally-facilitated, manual annotation of immune response genes provided expert-level curation of  <dig>  gene models and  <dig>  transcripts, of which  <dig>  annotated using sequence available only from other organisms.
this extensive annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type  <dig> interferon families.
we found gene duplications for  <dig> genes, including  <dig> immune response genes and five non-immune response genes discovered in the annotation process.
we used a functional approach to discover and accurately annotate porcine immune response genes.
using co-expression analysis of transcriptional profiling data from studies on blood, macrophages, as well as lymph nodes, we identified a large cluster  that exhibited a correlated positive response upon infection across a number of pathogens or due to different immune stimuli.
interestingly, this gene cluster  is enriched for known general human immune response genes  <cit> , yet only 16% of these genes have been annotated as immune response in the gene ontology project.
overall this approach has provided new putative immune response annotation for over  <dig> porcine genes.
a phylogenetic analysis of the encoded proteins of cluster  <dig> genes showed high rates of evolutionary change at the amino acid level, confirming the hypothesis that such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time.
these comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

 the co-evolution of host response and pathogen evasion mechanisms drives variation in the response to infectious diseases at individual, population and species levels, as well as at higher order taxonomic units. immunity-related genes are “tailored to the niche that a species occupies” and exhibit many features of positive selection, including polygeny and clustering of loci, high rates of non-synonymous substitutions, allele and gene conversion, generation of repertoires, rapid evolution, co-evolution, association with diseases and networking. the host response to a pathogen requires concerted action of a huge set of immunity-related genes recently referred to as the immunome. the immunome was first defined as the totality of rearranged antibody and antigen receptor genes present in all living individuals of a species, including variations in the somatic rearrangements. that definition was further adapted to describe the whole set of genes related to both innate and acquired immunity as identified from whole genome sequencing and functional genomics studies. the progress in genome sequencing of human, model and non-model animal species, including livestock species, now permits comparative analysis across many species. such studies have highlighted the divergence in innate immune responses between humans and mice, the most widely-studied experimental animal model. the domestic pig, in the suidae family within the cetartiodactyl order of eutherian mammals, has been used a model in medical research due to its similarity to humans in size and physiology, including comparable digestive, respiratory, and immune systems. the cetartiodactyl order first appeared 60- million years ago, while the divergence for rodents and primates dates from and million years ago, respectively. primarily as a consequence of the fast sequence evolutionary rate of mouse, the pig has remained considerably more similar at the dna sequence level to humans. however, some immunological features are exceptional in the pig compared to humans and mice. pigs have an inverted lymph node structure and an unusual route for lymphocyte circulation. there are relatively high numbers of extra-thymic cd4/cd double positive t-cells and resting t-cells expressing sla class ii molecules. pigs can have high numbers of natural killer cells and γδ cells, harbor an unusual diversity of b-cell and antibody repertoire development, and have highly heritable variation in immune cell parameters. in pigs as in many other species, the numerous in vivo and in vitro studies on host-pathogen interactions and immunity stimulation, are now often based on functional genomics approaches such as transcriptomic approaches. with such rapid accumulation of high-dimensional data on immune response, network models are becoming increasingly important in the interpretation of such experimental data. correlation networks based on immune response data not only permit the identification of common regulatory mechanisms through integration with promoter/flanking sequences, but also provide evidence that un-annotated genes are involved in immune response pathways. thus an important aspect of gene annotation is the integration of structural analysis of rnas and genomes with functional data on transcriptional response to pathogens and immune stimuli. the purpose of the immune response annotation group was to explore the porcine immunome by exploiting the recently available genome sequence assembly. a gene list for detailed manual gene annotation using otterlace was compiled using gene ontology annotation and literature sources. analyses combined structural, evolutionary and functional approaches. we report a refined gene structure annotation on greater than genes involved in immunity; data on positive selection pressure of a subset of the proteins predicted to be encoded by these genes; and a correlation network analysis of transcriptomic data from various disease and immunological models. these three levels of data contribute to a better characterization of the pig immunome and provide a comparative genomic appraisal across mammals.  extensive manual annotation of the genomic complement of porcine immune response genes the immune response annotation group members used otterlace to manually annotate over loci in porcine build selected based on their membership in immune response processes or gene ontology immune response annotation. the go term used as an inclusion criterion was “immune system process”; go: these structural annotations were transferred over to build and discrepancies were addressed to determine the final annotation results. members confirmed automated annotation of known genes through manual annotation of transcripts and gene models; annotated transcripts contained the full-length protein coding sequence. twenty-six pseudogenes were also identified during the annotation. *number of gene objects created in the otterlace annotation system. ** processed and non-processed pseudogenes are included. # no porcine est or cdna sequence was available to create these transcript predictions. importantly, the cross-species alignment tools in otterlace allowed annotation of transcripts in the pig genome using only mrna sequence from other species. such transcripts without specific porcine sequence support were often made using human cdnas, as there are many more human than pig sequences in the databases. the conservation between human and pig in terms of synteny is three times greater than between human and mouse, and the pig is more closely-related at the dna sequence level to humans than either is to the mouse. specifically for immune gene families, recent analysis at the cdna sequence level of pig, mouse and human has shown that the great majority of human genes that were lost through evolution in the mouse were retained in the pig. conversely, very few mouse genes that were lost through evolution in the human were found in the pig. comparison of expansion or contraction of orthologous gene families indicated far more similar rates and classes of genes in humans and pigs than in mice. the conservation of homology and structural motifs of unambiguous orthologs from pigs, mice and humans revealed that the overall mean similarity to human proteins was significantly higher for pigs compared to mouse. the following sections provide summaries of important groups of genes for which the manual annotation revealed new insight. one important region highly relevant to immune response, the swine leukocyte antigen complex, has been previously annotated in detail and will not be discussed here. t cell receptor genes in the tcr complex possess highly repetitive sequences, so that it is difficult to generate correctly reconstructed loci from shotgun sequencing with low redundancy or short-read next-generation sequencing. therefore, intensive sequencing efforts were applied to the tra/trd and trb loci. the pig trd locus is embedded in tra, and d and j segments, and genes encoding the c region of tcr δ are located between the v segments of tcr α/δ and j genes of tcr α, as observed in other mammals. all of the human jα segments correspond to those of pig, and most of mouse jα can be allocated to orthologs in pig. these indicate functional similarity of the tcr α molecule between human, mouse and pig. on the other hand, the pig tcr δ gene has a more complicated structure than those of human and mouse. pig has at least dδ genes, while human and mouse have and respectively. the pig dδ genes are frequently used in functional tcr δ transcripts with up to concatenated domains. thus, the pig can generate a high diversity of tcr δ chain molecules to cope with antigens, which may be related to the fact that the percentage of γδ t cells in peripheral blood is much higher in pig than in human and mouse. as for trb, pig has functional dβ-jβ-cβ units, while human and mouse each have units. immunoglobulin ighv gene diversity is highly restricted, as in cattle, but all known porcine ighv genes belong to a single family, ighv whereas all cattle ighv belong to ighv. the lambda light chain locus on ssc contains iglv gene segments, with appearing functional. the locus is organized into two distinct clusters, a constant -proximal cluster containing iglv family members, and a c-distal cluster containing iglv and iglv family members. the porcine iglv subgroup genes have recently expanded, suggesting a particularly effective role in immunity to porcine-specific pathogens, especially since iglv expression is nearly exclusively restricted to the iglv and iglv. the c-distal iglv cluster also contains three non-functional iglv family members that are orthologous to iglv that are exclusively expressed in cattle. the igl locus contains three tandem iglj–iglc cassettes, two of which are functional, and a fourth iglj with no corresponding iglc. the kappa light chain locus on ssc is comprised of at least igkv genes, of which are functional and belong to either the igkv or igkv gene families, five igkj genes that lie kb downstream, and a single igkc gene. polymorphisms within the individual duroc sow that was genome sequenced revealed alleles that differed by as much as percent among igkv genes and as much as eight percent in amino acid sequence among iglv genes. the porcine immunoglobulin genes have evolved such that specific gene families have expanded and contracted with respect to other species, notably cattle. the high level of allelic variation found within the antibody light chain loci substantially expands the population diversity of the porcine antibody repertoire. in the kappa locus, in particular, many igkv family members share specific parts of coding regions, such as complementary determining region between genes but not between alleles. thus, germline gene conversion may provide a mechanistic basis for the high level of igkv allelic variation. killer immunoglobulin-like receptor pigs appear to have a single kir gene in contrast to cattle, horses and primates in which there is an expanded kir gene family. in rodents, the functionally equivalent receptors are encoded by the expanded gene family of killer cell lectin-like receptor genes of which klra has paralogues whilst pigs have a single ly gene with two putative orthologs. the limited nk cell repertoire in pigs is not linked with any deficiency in nk cell numbers but there is evidence for a connection between high variability between individual animals and performance under low health status conditions. one would anticipate, given the function of nk cell receptors in recognition of mhc class proteins, that there must be some unique feature to this interaction in the pig to allow nk cells to function irrespective of unlinked polymorphism and the sla loci. immune gene family expansion a preliminary analysis of immune gene families has previously compared humans, mice and pigs. in the current analysis, artiodactyl-associated families were also included based upon expansions noted in the bovine genome. our porcine genome analyses show that some of these expansions are also present in the porcine genome, indicating an artiodactyl-specific expansion. other expansions are not present in the pig genome, providing additional support for a ruminant-specific expansion. results are summarized in table  and full details shown in additional file 1: table s * numbers of confirmed pseudogenes are shown in parentheses. agp and dcr: asialo- glycoprotein and dc receptor; mc: mast cell; sp: serine protease; up: ubiquitin-protein. the cathelicidin gene family was expanded, with genes compared to only one in human and mouse. the expansion appears to be artiodactyl-specific since cattle also have genes. thirty-four beta defensin genes were detected in the swine genome assembly, similar to the human genome, but substantially less than the > beta-defensin genes reported from cattle. a recent report annotated porcine defensins in the high-throughput genome sequences pre-assembly, which indicates that our annotation of for the current genome assembly adds to the previous annotation of this family. for this work, we tested the procedure based on hidden-markov model that was used in the bovine genome project, but primarily used our curation protocol established for genome-wide annotation of beta-defensin families in the human, chimpanzee, mouse, rat, and dog. as a more sensitive procedure, hmm could overestimate the functional gene numbers without manual curation. thus far, we suggest that the composition of the porcine beta-defensin family is more similar to human than bovine. a similar result is observed for the c-type lysozyme family in pigs, which has genes, while and are found in the bovine, human and mouse genomes, respectively. thus, our analysis of the second artiodactyl genome indicates that beta-defensin and c-type lysozyme family expansions observed in cattle may be ruminant-specific adaptations. because pigs are omnivores and cows are herbivores, it is tempting to speculate that these differences may be due to different exposure to gut microbiota. pigs have type i interferon genes, twice the number in human, as well as pseudogenes. cattle have type i ifn genes. this expansion is focused on interferon subtypes ifnw and ifnd; pigs and cattle share novel subtypes of ifnd, ifnt, ifnaw and many more isoforms of ifnw. thus, expansion of interferon genes is not ruminant-specific as previously proposed, although duplication within some specific sub-families appears to be either bovine- or pig-specific. data presented in table  represents an expanded analysis of the gene families that were presented in the recent porcine genome paper. four additional gene families, slam superfamily, granzyme/mast cell tryptase/serine protease superfamily, cytidine deaminase-like superfamily and immunity related guanosine triphosphatase superfamily are included. these new analyses reveal a slight expansion in the slam superfamily in cow and pig relative to human, a relatively large expansion in the granzyme/mast cell tryptase/serine protease superfamily in mouse and cow relative to humans and pigs, and an extremely large expansion in the immunity related guanosine triphosphatase superfamily in mouse relative to the other species. in contrast, the number of cytidine deaminase-like superfamily members is human is twice that found in pigs, mice and cows. the total number of pig, mouse and cow gene families that have undergone expansion of >25% of family members relative to human are and respectively. thus familial gene expansion in pigs relative to humans has occurred at half the rate of mice and cows. conversely, the total number of gene families in pig, mouse and cow that have undergone contraction of >25% of family members relative to human are and respectively. familial gene contraction in pigs relative to humans has occurred at roughly the same rate of mice and cows. an additional analysis of orthology preservation of familial gene expansion for the four species deriving from the family member expansion analysis revealed that 1: orthology conservation was found for of the genes. mice had the largest number of unique genes, more than twice the number found in cattle, and more than all of the others combined. in contrast, pigs have 11-, 5- and 2-fold fewer unique genes compared to the mouse, cow and human, respectively. pair-wise analysis indicates that pigs and cows share genes that are not found in humans or mice. these genes are members of the bpi superfamily, bpifb and bpifb9; cathelicidin superfamily cathl, cathl, cathl, cathl, cathl, cathl; ccl chemokine superfamily, ccl3l2; cd163/wc superfamily, loc100337197; clect superfamily, klrj and prg3l1; granzyme/mast cell tryptase/serine protease superfamily, gzmal, mcpt3; immunity related guanosine triphosphatase superfamily, irgcl1; nlr and pyrin superfamily, nlrp12l; and rnase a family, brb and loc. furthermore, analysis of three species at a time indicates that humans and pigs share genes that are not found in mice; mice and humans share genes that are not found in pigs; and mice and pigs share genes that are not found in humans. these conclusions must be tempered with the observation that the porcine genome is still incomplete and additional family members may be discovered. indeed, in the course of looking for known genes in the current porcine genome build, we identified genes that do not appear in the build assembly; please see additional file 2: table s however, these similarities, especially for pig and human, further reinforce the use of the pig as a large animal model of the immune response of humans. gene duplication in the course of annotating the immune response gene families shown in table  we found indication of gene duplication and pseudogenes in the build assembly. a summary of putative true gene duplications is shown in additional file 3: table s using extreme sequence similarity as a metric, many duplications appear to be due to assembly artifacts. these artifactually duplicated genes fall into different categories: on the same chromosome and mapped to the same assembly scaffold, on the same chromosome and mapped to a different assembly scaffold, and on different chromosomes. the duplications that appear on different chromosomes are especially problematic because of the need to assure bac specificity. one gene, tnfrsf10a, which is listed provisionally here as artifactually duplicated, proved especially problematic since there is equal evidence for both artifactual and true duplication; directed studies will be necessary to determine the nature of this duplication. evidence for the true duplication of immune-related genes: atf cd cd cd crp, ddx3x, gstp gzma, ifit il1b, irgc, itln and oas and non-immune genes, appears unequivocal. interestingly, the il1b gene duplication, in which evidence for a partial duplication had been reported, is unique in mammals. predicted proteins expressed from the il1b gene and its duplicate are both aa in length, but only 86% identical. further, their mrnas have different expression patterns in adult and embryonic tissues, and different responses to endotoxin in macrophages. unigene expression profiling of other true duplicated genes indicates that they are differentially expressed. for example the macrophage scavenger receptor cd is expressed primarily in adipose tissue, blood, lung, mammary gland, and ovary, whereas the porcine-restricted duplicated gene with an unknown function, cd68l, has a wider tissue distribution with expression in adipose tissue, adrenal gland, blood, cartilage, heart, intestine, lung, lymph node, muscle, ovary, placenta, skin, spleen, thymus and trachea. the pattern recognition receptor cd is highly expressed primarily in adipose tissue, heart, mammary gland and muscle. the truncated protein cd36l, with an unknown function, is also duplicated in the cow and is expressed at lower levels in blood, bone marrow liver, lung and mammary gland. the differential expression patterns of these genes support their phylogenetic and functional divergence. functional annotation of immune genes: finding genes with immune response patterns similar to known immune system genes co-expression of genes can be used to provide evidence for membership in specific processes, such as immune response, when a substantial proportion of the members of an expression cluster have similar functions. we collected all data reported for the 24k affymetrix genechip for experiments with an immune component such as infection of tissues or cells with bacteria, viruses, or stimulation with lipopolysaccharide. using this targeted set of chips, which included public data as well as several un-published datasets from our groups, we calculated a within-group correlation for each experimental dataset. this approach emphasizes the response to pathogen/stimulus. these correlations were then used in the co-expression network tool biolayout express3d to generate and visualize a transcriptome network for porcine generic immune response. using this correlation network, we then identified clusters with go term annotation enrichment related to immune response. significance of enrichment was determined as described in methods. in additional file 5: table s and additional file 6: table s, we document these results. cluster with probesets representing at least transcripts, was significantly enriched for many go terms relevant to immune response pathways, including type i interferon-mediated signaling pathway and cytokine-mediated signaling pathway, as well as response to virus, and proteasome core complex. cluster is highlighted in figure  and includes many immune-related genes such as il jak irf irf irf ifit ifit ifit cd cd cd many stat, psmb and casp gene family members, mx mx cxcl ccrl wars, slc11a and complement genes c1r, c1s, and c of these probesets, are annotated with the go term originally used as a major criterion for the irag gene list, which is a -fold enrichment. this cluster is also three-fold enriched for porcine orthologs of common immune response genes identified by jenner and young based on a meta-analysis of microarray data from a number of pathogens/ immune stimulations of several human cell types. thus cluster clearly is enriched for a large number of genes likely to be involved in the immune response of swine. importantly, most of these probesets are not already annotated as immune response genes, as only 16% have the go: term annotation. further, the average rna level for these probesets across the immune response datasets is shown in figure  the pattern is clearly one of activation upon infection or treatment, as global increases in expression levels are seen in many datasets upon immune stimulation. thus this correlation analysis provides evidence for the involvement of many new genes in the porcine immune response. a second cluster with enrichment of go: is cluster. as with cluster cluster is also enriched for jenner-young ir genes as well as genes manually annotated as “innate immune” genes in innatedb. for the global go annotation, only anti-apoptosis was found significantly enriched. genes in this smaller cluster of members include several negative regulators of innate/inflammatory pathways. the pattern of expression of these genes is similar to that of cluster with cluster genes clearly activated in lps-stimulated macrophages and in lymph nodes or blood from pigs infected with salmonella. interestingly, in contrast to cluster several datasets did not show activation of genes represented by the probesets in cluster genes on average, including lymph nodes infected with pcv or prrsv. a third cluster contains probesets, containing probesets representing several t-cell related genes as well as other immunity genes such as il7r, gzmb and stat this cluster appears to document gene expression specific to t cells and neutrophils, as expression is detected in lymph node and whole blood datasets, and absent in macrophages. the global annotation identified regulation of immune response as well as several t cell receptor terms as enriched in this cluster. the pattern of expression is not clearly correlated with response to immune stimulation, which is consistent with the observation that these genes are not enriched for j-y ir or innatedb genes, but are enriched for genes with go: annotation. finally, cluster is enriched only for probesets annotated with the go: term, and shows early and robust induction in macrophages infected with prrsv and in lymph nodes in animals infected with salmonella enterica serovar typhimurium, but variable low to modest induction in macrophages by lps and other challenges. this cluster of probesets includes a number representing mhc genes as well as cfp, cyba, ly and irf comprehensive go annotation shows this cluster is enriched for interferon gamma-mediated signaling pathway, t cell receptor signaling pathway, cytokine-mediated signaling pathway, and immune response genes. the established gene lists for generic immune responses at the level of the transcriptome can be used to improve the annotation of a large number of genes/transcripts in the porcine genome related to immune response. especially for cluster which is significantly enriched for probesets annotated as human immune response genes, these data provide foundational information that can be used for human-pig comparisons at several levels. for example, global comparisons of promoter sequence elements between pig and other species can be performed using draft genome assembly information, as shown recently for an analysis of the cyp27b gene promoter. previously, substitution of human orthologous promoters was useful in prediction of sets of nfκb target genes in the pig. given the recent expansion of transcriptomic datasets on immune response, especially for those analyzing response to infection of specific tissues or cell types, the pig genome will now be invaluable in bioinformatic approaches to recognize known and novel regulatory motifs in immune response genes. prior annotation as immune response genes, as demonstrated herein, will provide further confidence for genes clustered by their transcriptomic response to an immune stimulation. an accelerated rate of evolution for immunity-related genes as recently reported, an analysis of predicted rates of evolutionary change was carried out on a randomly selected subset of immunity-related pig proteins from the irag annotated gene set. this analysis showed rates of positive selection between and %, depending on the analysis method. to confirm and extend this significant increase in the rate of positive selection for immunity-related genes in swine, we analyzed the proteins present in the cluster found in the above analysis to be significantly enriched for probesets annotated as immunity-related genes. a set of proteins was analyzed with the phyleasprog web server, and a subset of proteins having at least orthologs and being compatible with threshold of statistical significance was included in final analyses. among these proteins, proteins are under positive selection with q <, and with q <; i.e. 15% and 17% respectively. thus, cluster is as rich in positively selected proteins, as was the subset of immunity-related pig proteins recently published in the swine genome sequence paper. at the whole genome level, it has been reported that the rate of positive selection, computed on different types of data and different methods, is % in human, % in chimpanzee, close to 5% in cattle, dog and horse, and % in pig. our results show a significant increase in the rate of positive selection for immunity-related genes in swine. these results confirm that positively selected genes in swine are enriched for roles in defense and immunity in mammals, as already shown in human, cow, five other mammals, as well as birds like the zebra finch. other functions are also reported as privileged targets for an accelerated evolutionary rate of related genes in mammals, such as reproduction, taste perception, chemosensory reception, and olfaction, as recently shown in pig. by branch-site analysis, we detected an accelerated evolution of several amino acids specific to pig in proteins, including sppl2a, jak ppp2r5c, chd- tspan nmt gbp hexb, fam26f, lman2l, ankmy phf20l ddx pde8a, lcp usp slc24a. the projections of amino acids under positive selection onto 3d structure of the four proteins casp hexb, gbp and ppp2r5c are shown via phyleasprog web server. the ppp25rc protein is known as the protein phosphatase regulatory subunit b’, gamma and is amino acids long. within a segment of amino acids from position to a total of amino acids were found under positive selection. conversely, amino acids were found under purifying selection from position to. the interproscan revealed matches with the protein phosphatase 2a, regulatory b subunit, b, and an armadillo-type fold from position to by the superfamily method. these striking results suggest two potentially distinct subdomains for the protein ppp25rc in swine. the hexb protein, hexosaminidase b, is amino acids long in swine. hexosaminidase b is the beta subunit of the lysosomal enzyme beta-hexosaminidase that, together with the cofactor gm activator protein, catalyzes the degradation of the ganglioside gm and other molecules containing terminal n-acetylhexosamines. two amino acids were found under positive selection at positions and both amino acids map to the glycoside hydrolase catalytic domain. at position the amino acid maps also to a beta-hexosaminidase subunit related to beta-n-acetylhexosaminidase activity. the casp protein, known as the caspase apoptosis-related cysteine peptidase comprises amino acids in swine and has also been found under accelerated evolution rate in human. two amino acids were found under positive selection at positions and the amino acid at position specifically maps to a predicted domain referred to as domain peptidase c caspase precursor p core-il1bcenzyme. the gbp protein is known as the guanylate binding protein interferon-inducible and is amino acids long. a unique amino acid has been found under positive selection at position in the guanylate binding protein, c-terminal domain. the availability of whole genome sequences has paved the way for renewed approaches to study the molecular signatures of natural selection at unprecedented scales. in addition, amino acids found under positive selection are highly interesting candidates to target for further biological analyses and understanding of the link between structures and functions. in genetic studies aimed at identifying nucleotide polymorphisms involved in the variation of target traits, the analysis of evolutionary constraints at candidate mutations should provide a fundamental, additional layer of information.  our computationally-facilitated, manual annotation of immune response genes provided expert-level curation of gene models and transcripts, of which annotated using sequence available only from other organisms. this extensive annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type interferon families. we found gene duplications for genes, including immune response genes and five non-immune response genes discovered in the annotation process. we used a functional approach to discover and accurately annotate porcine immune response genes. using co-expression analysis of transcriptional profiling data from studies on blood, macrophages, as well as lymph nodes, we identified a large cluster that exhibited a correlated positive response upon infection across a number of pathogens or due to different immune stimuli. interestingly, this gene cluster is enriched for known general human immune response genes, yet only 16% of these genes have been annotated as immune response in the gene ontology project. overall this approach has provided new putative immune response annotation for over porcine genes. a phylogenetic analysis of the encoded proteins of cluster genes showed high rates of evolutionary change at the amino acid level, confirming the hypothesis that such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. these comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response. 
a co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. these comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response. our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over porcine genes. we used a functional approach to discover and accurately annotate porcine immune response genes. our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. we found gene duplications for genes, including immune response genes and five non-immune response genes discovered in the annotation process. the immune response annotation group used computational curation and manual annotation of the swine genome assembly to refine the currently available automated annotation of immunity-related genes through sequence-based comparison to genes in other species. interestingly, this gene cluster is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. the domestic pig is known as an excellent model for human immunology and the two species share many pathogens. susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. the completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. manual annotation provided evidence for many new alternative splice variants and gene duplications. this extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type interferon families. immune responseporcinegenome annotationco-expression networkphylogenetic analysisaccelerated evolution a phylogenetic analysis of the encoded proteins of cluster genes showed that 15% exhibited an accelerated evolution as compared to % across the entire genome.
 the domestic pig is known as an excellent model for human immunology and the two species share many pathogens. susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. the completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.  the immune response annotation group used computational curation and manual annotation of the swine genome assembly to refine the currently available automated annotation of immunity-related genes through sequence-based comparison to genes in other species. within these genes, we annotated transcripts. annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type interferon families. we found gene duplications for genes, including immune response genes and five non-immune response genes discovered in the annotation process. manual annotation provided evidence for many new alternative splice variants and gene duplications. over transcripts without porcine sequence evidence were detected using cross-species annotation. we used a functional approach to discover and accurately annotate porcine immune response genes. a co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. interestingly, this gene cluster is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. a phylogenetic analysis of the encoded proteins of cluster genes showed that 15% exhibited an accelerated evolution as compared to % across the entire genome.  this extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over porcine genes. our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. these comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response. immune responseporcinegenome annotationco-expression networkphylogenetic analysisaccelerated evolution 