Variations in antibody repertoires correlate with vaccine responses.

An important challenge in vaccine development is to figure out why a vaccine succeeds in some individuals and fails in others. Although antibody repertoires hold the key to answering this question, there have been very few personalized immunogenomics studies so far aimed at revealing how variations in immunoglobulin genes affect a vaccine response. We conducted an immunosequencing study of 204 calves vaccinated against bovine respiratory disease (BRD) with the goal to reveal variations in immunoglobulin genes and somatic hypermutations that impact the efficacy of vaccine response. Our study represents the largest longitudinal personalized immunogenomics study reported to date across all species, including humans. To analyze the generated data set, we developed an algorithm for identifying variations of the immunoglobulin genes (as well as frequent somatic hypermutations) that affect various features of the antibody repertoire and titers of neutralizing antibodies. In contrast to relatively short human antibodies, cattle have a large fraction of ultralong antibodies that have opened new therapeutic opportunities. Our study reveals that ultralong antibodies are a key component of the immune response against the costliest disease of beef cattle in North America. The detected variants of the cattle immunoglobulin genes, which are implicated in the success/failure of the BRD vaccine, have the potential to direct the selection of individual cattle for ongoing breeding programs.

Assembly of a pangenome for global cattle reveals missing sequences and novel structural variations, providing new insights into their diversity and evolutionary history.

A cattle pangenome representation was created based on the genome sequences of 898 cattle representing 57 breeds. The pangenome identified 83 Mb of sequence not found in the cattle reference genome, representing 3.1% novel sequence compared with the 2.71-Gb reference. A catalog of structural variants developed from this cattle population identified 3.3 million deletions, 0.12 million inversions, and 0.18 million duplications. Estimates of breed ancestry and hybridization between cattle breeds using insertion/deletions as markers were similar to those produced by single nucleotide polymorphism-based analysis. Hundreds of deletions were observed to have stratification based on subspecies and breed. For example, an insertion of a Bov-tA1 repeat element was identified in the first intron of the APPL2 gene and correlated with cattle breed geographic distribution. This insertion falls within a segment overlapping predicted enhancer and promoter regions of the gene, and could affect important traits such as immune response, olfactory functions, cell proliferation, and glucose metabolism in muscle. The results indicate that pangenomes are a valuable resource for studying diversity and evolutionary history, and help to delineate how domestication, trait-based breeding, and adaptive introgression have shaped the cattle genome.

Long read isoform sequencing reveals hidden transcriptional complexity between cattle subspecies.

The Iso-Seq method of full-length cDNA sequencing is suitable to quantify differentially expressed genes (DEGs), transcripts (DETs) and transcript usage (DTU). However, the higher cost of Iso-Seq relative to RNA-seq has limited the comparison of both methods. Transcript abundance estimated by RNA-seq and deep Iso-Seq data for fetal liver from two cattle subspecies were compared to evaluate concordance. Inter-sample correlation of gene- and transcript-level abundance was higher within technology than between technologies. Identification of DEGs between the cattle subspecies depended on sequencing method with only 44 genes identified by both that included 6 novel genes annotated by Iso-Seq. There was a pronounced difference between Iso-Seq and RNA-seq results at transcript-level wherein Iso-Seq revealed several magnitudes more transcript abundance and usage differences between subspecies. Factors influencing DEG identification included size selection during Iso-Seq library preparation, average transcript abundance, multi-mapping of RNA-seq reads to the reference genome, and overlapping coordinates of genes. Some DEGs called by RNA-seq alone appear to be sequence duplication artifacts. Among the 44 DEGs identified by both technologies some play a role in immune system, thyroid function and cell growth. Iso-Seq revealed hidden transcriptional complexity in DEGs, DETs and DTU genes between cattle subspecies previously missed by RNA-seq.

metaFlye: scalable long-read metagenome assembly using repeat graphs.

Long-read sequencing technologies have substantially improved the assemblies of many isolate bacterial genomes as compared to fragmented short-read assemblies. However, assembling complex metagenomic datasets remains difficult even for state-of-the-art long-read assemblers. Here we present metaFlye, which addresses important long-read metagenomic assembly challenges, such as uneven bacterial composition and intra-species heterogeneity. First, we benchmarked metaFlye using simulated and mock bacterial communities and show that it consistently produces assemblies with better completeness and contiguity than state-of-the-art long-read assemblers. Second, we performed long-read sequencing of the sheep microbiome and applied metaFlye to reconstruct 63 complete or nearly complete bacterial genomes within single contigs. Finally, we show that long-read assembly of human microbiomes enables the discovery of full-length biosynthetic gene clusters that encode biomedically important natural products.

Colonialism in South Africa leaves a lasting legacy of reduced genetic diversity in Cape buffalo.

The iconic Cape buffalo has experienced several documented population declines in recent history. These declines have been largely attributed to the late 19th century rinderpest pandemic. However, the effect of the rinderpest pandemic on their genetic diversity remains contentious, and other factors that have potentially affected this diversity include environmental changes during the Pleistocene, range expansions and recent human activity. Motivated by this, we present analyses of whole genome sequencing data from 59 individuals from across the Cape buffalo range to assess present-day levels of genome-wide genetic diversity and what factors have influenced these levels. We found that the Cape buffalo has high average heterozygosity overall (0.40%), with the two southernmost populations having significantly lower heterozygosity levels (0.33% and 0.29%) on par with that of the domesticated water buffalo (0.29%). Interestingly, we found that these lower levels are probably due to recent inbreeding (average fraction of runs of homozygosity 23.7% and 19.9%) rather than factors further back in time during the Pleistocene. Moreover, detailed investigations of recent demographic history show that events across the past three centuries were the main drivers of the exceptional loss of genetic diversity in the southernmost populations, coincident with the onset of colonialism in the southern extreme of the Cape buffalo range. Hence, our results add to the growing body of studies suggesting that multiple recent human-mediated impacts during the colonial period caused massive losses of large mammal abundance in southern Africa.

Gaur genome reveals expansion of sperm odorant receptors in domesticated cattle.

BACKGROUND: The gaur (Bos gaurus) is the largest extant wild bovine species, native to South and Southeast Asia, with unique traits, and is listed as vulnerable by the International Union for Conservation of Nature (IUCN). RESULTS: We report the first gaur reference genome and identify three biological pathways including lysozyme activity, proton transmembrane transporter activity, and oxygen transport with significant changes in gene copy number in gaur compared to other mammals. These may reflect adaptation to challenges related to climate and nutrition. Comparative analyses with domesticated indicine (Bos indicus) and taurine (Bos taurus) cattle revealed genomic signatures of artificial selection, including the expansion of sperm odorant receptor genes in domesticated cattle, which may have important implications for understanding selection for male fertility. CONCLUSIONS: Apart from aiding dissection of economically important traits, the gaur genome will also provide the foundation to conserve the species.

Defining the caprine γδ T cell WC1 multigenic array and evaluation of its expressed sequences and gene structure conservation among goat breeds and relative to cattle.

Workshop cluster 1 (WC1) molecules are part of the scavenger receptor cysteine-rich (SRCR) superfamily and act as hybrid co-receptors for the γδ T cell receptor and as pattern recognition receptors for binding pathogens. These members of the CD163 gene family are expressed on γδ T cells in the blood of ruminants. While the presence of WC1+ γδ T cells in the blood of goats has been demonstrated using monoclonal antibodies, there was no information available about the goat WC1 gene family. The caprine WC1 multigenic array was characterized here for number, structure and expression of genes, and similarity to WC1 genes of cattle and among goat breeds. We found sequence for 17 complete WC1 genes and evidence for up to 30 SRCR a1 or d1 domains which represent distinct signature domains for individual genes. This suggests substantially more WC1 genes than in cattle. Moreover, goats had seven different WC1 gene structures of which 4 are unique to goats. Caprine WC1 genes also had multiple transcript splice variants of their intracytoplasmic domains that eliminated tyrosines shown previously to be important for signal transduction. The most distal WC1 SRCR a1 domains were highly conserved among goat breeds, but fewer were conserved between goats and cattle. Since goats have a greater number of WC1 genes and unique WC1 gene structures relative to cattle, goat WC1 molecules may have expanded functions. This finding may impact research on next-generation vaccines designed to stimulate γδ T cells.

Adaptive selection signatures in river buffalo with emphasis on immune and major histocompatibility complex genes.

River buffalo is an agriculturally important species with many traits, such as disease tolerance, which promote its use worldwide. Highly contiguous genome assemblies of the river buffalo, goat, pig, human and two cattle subspecies were aligned to study gene gains and losses and signs of positive selection. The gene families that have changed significantly in river buffalo since divergence from cattle play important roles in protein degradation, the olfactory receptor system, detoxification and the immune system. We used the branch site model in PAML to analyse single-copy orthologs to identify positively selected genes that may be involved in skin differentiation, mammary development and bone formation in the river buffalo branch. The high contiguity of the genomes enabled evaluation of differences among species in the major histocompatibility complex. We identified a Babesia-like L1 LINE insertion in the DRB1-like gene in the river buffalo and discuss the implication of this finding.

Distinctive gene expression patterns and imprinting signatures revealed in reciprocal crosses between cattle sub-species.

BACKGROUND: There are two genetically distinct subspecies of cattle, Bos taurus taurus and Bos taurus indicus, which arose from independent domestication events. The two types of cattle show substantial phenotypic differences, some of which emerge during fetal development and are reflected in birth outcomes, including birth weight. We explored gene expression profiles in the placenta and four fetal tissues at mid-gestation from one taurine (Bos taurus taurus; Angus) and one indicine (Bos taurus indicus; Brahman) breed and their reciprocal crosses. RESULTS: In total 120 samples were analysed from a pure taurine breed, an indicine breed and their reciprocal cross fetuses, which identified 6456 differentially expressed genes (DEGs) between the two pure breeds in at least one fetal tissue of which 110 genes were differentially expressed in all five tissues examined. DEGs shared across tissues were enriched for pathways related to immune and stress response functions. Only the liver had a substantial number of DEGs when reciprocal crossed were compared among which 310 DEGs were found to be in common with DEGs identified between purebred livers; these DEGs were significantly enriched for metabolic process GO terms. Analysis of DEGs across purebred and crossbred tissues suggested an additive expression pattern for most genes, where both paternal and maternal alleles contributed to variation in gene expression levels. However, expression of 5% of DEGs in each tissue was consistent with parent of origin effects, with both paternal and maternal dominance effects identified. CONCLUSIONS: These data identify candidate genes potentially driving the tissue-specific differences between these taurine and indicine breeds and provide a biological insight into parental genome effects underlying phenotypic differences in bovine fetal development.

Variants at the ASIP locus contribute to coat color darkening in Nellore cattle.

BACKGROUND: Nellore cattle (Bos indicus) are well-known for their adaptation to warm and humid environments. Hair length and coat color may impact heat tolerance. The Nellore breed has been strongly selected for white coat, but bulls generally exhibit darker hair ranging from light grey to black on the head, neck, hump, and knees. Given the potential contribution of coat color variation to the adaptation of cattle populations to tropical and sub-tropical environments, our aim was to map positional and functional candidate genetic variants associated with darkness of hair coat (DHC) in Nellore bulls. RESULTS: We performed a genome-wide association study (GWAS) for DHC using data from 432 Nellore bulls that were genotyped for more than 777 k single nucleotide polymorphism (SNP) markers. A single major association signal was detected in the vicinity of the agouti signaling protein gene (ASIP). The analysis of whole-genome sequence (WGS) data from 21 bulls revealed functional variants that are associated with DHC, including a structural rearrangement involving ASIP (ASIP-SV1). We further characterized this structural variant using Oxford Nanopore sequencing data from 13 Australian Brahman heifers, which share ancestry with Nellore cattle; we found that this variant originates from a 1155-bp deletion followed by an insertion of a transposable element of more than 150 bp that may impact the recruitment of ASIP non-coding exons. CONCLUSIONS: Our results indicate that the variant ASIP sequence causes darker coat pigmentation on specific parts of the body, most likely through a decreased expression of ASIP and consequently an increased production of eumelanin.

A Reference Genome Assembly of Simmental Cattle, Bos taurus taurus.

Genomics research has relied principally on the establishment and curation of a reference genome for the species. However, it is increasingly recognized that a single reference genome cannot fully describe the extent of genetic variation within many widely distributed species. Pangenome representations are based on high-quality genome assemblies of multiple individuals and intended to represent the broadest possible diversity within a species. A Bovine Pangenome Consortium (BPC) has recently been established to begin assembling genomes from more than 600 recognized breeds of cattle, together with other related species to provide information on ancestral alleles and haplotypes. Previously reported de novo genome assemblies for Angus, Brahman, Hereford, and Highland breeds of cattle are part of the initial BPC effort. The present report describes a complete single haplotype assembly at chromosome-scale for a fullblood Simmental cow from an F1 bison-cattle hybrid fetus by trio binning. Simmental cattle, also known as Fleckvieh due to their red and white spots, originated in central Europe in the 1830s as a triple-purpose breed selected for draught, meat, and dairy production. There are over 50 million Simmental cattle in the world, known today for their fast growth and beef yields. This assembly (ARS_Simm1.0) is similar in length to the other bovine assemblies at 2.86 Gb, with a scaffold N50 of 102 Mb (max scaffold 156.8 Mb) and meets or exceeds the continuity of the best Bos taurus reference assemblies to date.

A Reference Genome Assembly of American Bison, Bison bison bison.

Bison are an icon of the American West and an ecologically, commercially, and culturally important species. Despite numbering in the hundreds of thousands today, conservation concerns remain for the species, including the impact on genetic diversity of a severe bottleneck around the turn of the 20th century and genetic introgression from domestic cattle. Genetic diversity and admixture are best evaluated at genome-wide scale, for which a high-quality reference is necessary. Here, we use trio binning of long reads from a bison-Simmental cattle (Bos taurus taurus) male F1 hybrid to sequence and assemble the genome of the American plains bison (Bison bison bison). The male haplotype genome is chromosome-scale, with a total length of 2.65 Gb across 775 scaffolds (839 contigs) and a scaffold N50 of 87.8 Mb. Our bison genome is ~13× more contiguous overall and ~3400× more contiguous at the contig level than the current bison reference genome. The bison genome sequence presented here (ARS-UCSC_bison1.0) will enable new research into the evolutionary history of this iconic megafauna species and provide a new tool for the management of bison populations in federal and commercial herds.

Synaptogyrin-2 influences replication of Porcine circovirus 2.

Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association, ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (>24 hpi) and supernatant (>48hpi)(P < 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load.

Differences between predicted outer membrane proteins of genotype 1 and 2 Mannheimia haemolytica.

BACKGROUND: Mannheimia haemolytica strains isolated from North American cattle have been classified into two genotypes (1 and 2). Although members of both genotypes have been isolated from the upper and lower respiratory tracts of cattle with or without bovine respiratory disease (BRD), genotype 2 strains are much more frequently isolated from diseased lungs than genotype 1 strains. The mechanisms behind the increased association of genotype 2 M. haemolytica with BRD are not fully understood. To address that, and to search for interventions against genotype 2 M. haemolytica, complete, closed chromosome assemblies for 35 genotype 1 and 34 genotype 2 strains were generated and compared. Searches were conducted for the pan genome, core genes shared between the genotypes, and for genes specific to either genotype. Additionally, genes encoding outer membrane proteins (OMPs) specific to genotype 2 M. haemolytica were identified, and the diversity of their protein isoforms was characterized with predominantly unassembled, short-read genomic sequences for up to 1075 additional strains. RESULTS: The pan genome of the 69 sequenced M. haemolytica strains consisted of 3111 genes, of which 1880 comprised a shared core between the genotypes. A core of 112 and 179 genes or gene variants were specific to genotype 1 and 2, respectively. Seven genes encoding predicted OMPs; a peptidase S6, a ligand-gated channel, an autotransporter outer membrane beta-barrel domain-containing protein (AOMB-BD-CP), a porin, and three different trimeric autotransporter adhesins were specific to genotype 2 as their genotype 1 homologs were either pseudogenes, or not detected. The AOMB-BD-CP gene, however, appeared to be truncated across all examined genotype 2 strains and to likely encode dysfunctional protein. Homologous gene sequences from additional M. haemolytica strains confirmed the specificity of the remaining six genotype 2 OMP genes and revealed they encoded low isoform diversity at the population level. CONCLUSION: Genotype 2 M. haemolytica possess genes encoding conserved OMPs not found intact in more commensally prone genotype 1 strains. Some of the genotype 2 specific genes identified in this study are likely to have important biological roles in the pathogenicity of genotype 2 M. haemolytica, which is the primary bacterial cause of BRD.

New insights into mammalian sex chromosome structure and evolution using high-quality sequences from bovine X and Y chromosomes.

BACKGROUND: Mammalian X chromosomes are mainly euchromatic with a similar size and structure among species whereas Y chromosomes are smaller, have undergone substantial evolutionary changes and accumulated male specific genes and genes involved in sex determination. The pseudoautosomal region (PAR) is conserved on the X and Y and pair during meiosis. The structure, evolution and function of mammalian sex chromosomes, particularly the Y chromsome, is still poorly understood because few species have high quality sex chromosome assemblies. RESULTS: Here we report the first bovine sex chromosome assemblies that include the complete PAR spanning 6.84 Mb and three Y chromosome X-degenerate (X-d) regions. The PAR comprises 31 genes, including genes that are missing from the X chromosome in current cattle, sheep and goat reference genomes. Twenty-nine PAR genes are single-copy genes and two are multi-copy gene families, OBP, which has 3 copies and BDA20, which has 4 copies. The Y chromosome X-d1, 2a and 2b regions contain 11, 2 and 2 gametologs, respectively. CONCLUSIONS: The ruminant PAR comprises 31 genes and is similar to the PAR of pig and dog but extends further than those of human and horse. Differences in the pseudoautosomal boundaries are consistent with evolutionary divergence times. A bovidae-specific expansion of members of the lipocalin gene family in the PAR reported here, may affect immune-modulation and anti-inflammatory responses in ruminants. Comparison of the X-d regions of Y chromosomes across species revealed that five of the X-Y gametologs, which are known to be global regulators of gene activity and candidate sexual dimorphism genes, are conserved.

The Wnt Signaling Pathway Is Differentially Expressed during the Bovine Herpesvirus 1 Latency-Reactivation Cycle: Evidence That Two Protein Kinases Associated with Neuronal Survival, Akt3 and BMPR2, Are Expressed at Higher Levels during Latency.

Sensory neurons in trigeminal ganglia (TG) of calves latently infected with bovine herpesvirus 1 (BoHV-1) abundantly express latency-related (LR) gene products, including a protein (ORF2) and two micro-RNAs. Recent studies in mouse neuroblastoma cells (Neuro-2A) demonstrated ORF2 interacts with ß-catenin and a ß-catenin coactivator, high-mobility group AT-hook 1 (HMGA1) protein, which correlates with increased ß-catenin-dependent transcription and cell survival. ß-Catenin and HMGA1 are readily detected in a subset of latently infected TG neurons but not TG neurons from uninfected calves or reactivation from latency. Consequently, we hypothesized that the Wnt/ß-catenin signaling pathway is differentially expressed during the latency and reactivation cycle and an active Wnt pathway promotes latency. RNA-sequencing studies revealed that 102 genes associated with the Wnt/ß-catenin signaling pathway were differentially expressed in TG during the latency-reactivation cycle in calves. Wnt agonists were generally expressed at higher levels during latency, but these levels decreased during dexamethasone-induced reactivation. The Wnt agonist bone morphogenetic protein receptor 2 (BMPR2) was intriguing because it encodes a serine/threonine receptor kinase that promotes neuronal differentiation and inhibits cell death. Another differentially expressed gene encodes a protein kinase (Akt3), which is significant because Akt activity enhances cell survival and is linked to herpes simplex virus 1 latency and neuronal survival. Additional studies demonstrated ORF2 increased Akt3 steady-state protein levels and interacted with Akt3 in transfected Neuro-2A cells, which correlated with Akt3 activation. Conversely, expression of Wnt antagonists increased during reactivation from latency. Collectively, these studies suggest Wnt signaling cooperates with LR gene products, in particular ORF2, to promote latency.IMPORTANCE Lifelong BoHV-1 latency primarily occurs in sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. RNA sequencing studies revealed 102 genes associated with the Wnt/ß-catenin signaling pathway are differentially regulated during the latency-reactivation cycle. Two protein kinases associated with the Wnt pathway, Akt3 and BMPR2, were expressed at higher levels during latency but were repressed during reactivation. Furthermore, five genes encoding soluble Wnt antagonists and ß-catenin-dependent transcription inhibitors were induced during reactivation from latency. These findings are important because Wnt, BMPR2, and Akt3 promote neurogenesis and cell survival, processes crucial for lifelong viral latency. In transfected neuroblastoma cells, a viral protein expressed during latency (ORF2) interacts with and enhances Akt3 protein kinase activity. These findings provide insight into how cellular factors associated with the Wnt signaling pathway cooperate with LR gene products to regulate the BoHV-1 latency-reactivation cycle.

The antibody loci of the domestic goat (Capra hircus).

The domestic goat (Capra hircus) is an important ruminant species both as a source of antibody-based reagents for research and biomedical applications and as an economically important animal for agriculture, particularly for developing nations that maintain most of the global goat population. Characterization of the loci encoding the goat immune repertoire would be highly beneficial for both vaccine and immune reagent development. However, in goat and other species whose reference genomes were generated using short-read sequencing technologies, the immune loci are poorly assembled as a result of their repetitive nature. Our recent construction of a long-read goat genome assembly (ARS1) has facilitated characterization of all three antibody loci with high confidence and comparative analysis to cattle. We observed broad similarity of goat and cattle antibody-encoding loci but with notable differences that likely influence formation of the functional antibody repertoire. The goat heavy-chain locus is restricted to only four functional and nearly identical IGHV genes, in contrast to the ten observed in cattle. Repertoire analysis indicates that light-chain usage is more balanced in goats, with greater representation of kappa light chains (~ 20-30%) compared to that in cattle (~ 5%). The present study represents the first characterization of the goat antibody loci and will help inform future investigations of their antibody responses to disease and vaccination.

A high-quality annotated transcriptome of swine peripheral blood.

BACKGROUND: High throughput gene expression profiling assays of peripheral blood are widely used in biomedicine, as well as in animal genetics and physiology research. Accurate, comprehensive, and precise interpretation of such high throughput assays relies on well-characterized reference genomes and/or transcriptomes. However, neither the reference genome nor the peripheral blood transcriptome of the pig have been sufficiently assembled and annotated to support such profiling assays in this emerging biomedical model organism. We aimed to assemble published and novel RNA-seq data to provide a comprehensive, well-annotated blood transcriptome for pigs by integrating a de novo assembly with a genome-guided assembly. RESULTS: A de novo and a genome-guided transcriptome of porcine whole peripheral blood was assembled with ~162 million pairs of paired-end and ~183 million single-end, trimmed and normalized Illumina RNA-seq reads (~6 billion initial reads from 146 RNA-seq libraries) from five independent studies by using the Trinity and Cufflinks software, respectively. We then removed putative transcripts (PTs) of low confidence from both assemblies and merged the remaining PTs into an integrated transcriptome consisting of 132,928 PTs, with 126,225 (~95%) PTs from the de novo assembly and more than 91% of PTs spliced. In the integrated transcriptome, ~90% and 63% of PTs had significant sequence similarity to sequences in the NCBI NT and NR databases, respectively; 68,754 (~52%) PTs were annotated with 15,965 unique gene ontology (GO) terms; and 7618 PTs annotated with Enzyme Commission codes were assigned to 134 pathways curated by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Full exon-intron junctions of 17,528 PTs were validated by PacBio IsoSeq full-length cDNA reads from 3 other porcine tissues, NCBI pig RefSeq mRNAs and transcripts from Ensembl Sscrofa10.2 annotation. Completeness of the 5' termini of 37,569 PTs was validated by public cap analysis of gene expression (CAGE) data. By comparison to the Ensembl transcripts, we found that (1) the deduced precursors of 54,402 PTs shared at least one intron or exon with those of 18,437 Ensembl transcripts; (2) 12,262 PTs had both longer 5' and 3' termini than their maximally overlapping Ensembl transcripts; and (3) 41,838 spliced PTs were totally missing from the Sscrofa10.2 annotation. Similar results were obtained when the PTs were compared to the pig NCBI RefSeq mRNA collection. CONCLUSIONS: We built, validated and annotated a comprehensive porcine blood transcriptome with significant improvement over the annotation of Ensembl Sscrofa10.2 and the pig NCBI RefSeq mRNAs, and laid a foundation for blood-based high throughput transcriptomic assays in pigs and for advancing annotation of the pig genome.

The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation.

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination.

Whole genomic sequence analysis of Bacillus infantis: defining the genetic blueprint of strain NRRL B-14911, an emerging cardiopathogenic microbe.

BACKGROUND: We recently reported the identification of Bacillus sp. NRRL B-14911 that induces heart autoimmunity by generating cardiac-reactive T cells through molecular mimicry. This marine bacterium was originally isolated from the Gulf of Mexico, but no associations with human diseases were reported. Therefore, to characterize its biological and medical significance, we sought to determine and analyze the complete genome sequence of Bacillus sp. NRRL B-14911. RESULTS: Based on the phylogenetic analysis of 16S ribosomal RNA (rRNA) genes, sequence analysis of the 16S-23S rDNA intergenic transcribed spacers, phenotypic microarray, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we propose that this organism belongs to the species Bacillus infantis, previously shown to be associated with sepsis in a newborn child. Analysis of the complete genome of Bacillus sp. NRRL B-14911 revealed several virulence factors including adhesins, invasins, colonization factors, siderophores and transporters. Likewise, the bacterial genome encodes a wide range of methyl transferases, transporters, enzymatic and biochemical pathways, and insertion sequence elements that are distinct from other closely related bacilli. CONCLUSIONS: The complete genome sequence of Bacillus sp. NRRL B-14911 provided in this study may facilitate genetic manipulations to assess gene functions associated with bacterial survival and virulence. Additionally, this bacterium may serve as a useful tool to establish a disease model that permits systematic analysis of autoimmune events in various susceptible rodent strains.