John C. Schwartz

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ∼400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.

Single-molecule sequencing and conformational capture enable de novo mammalian reference genomes

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus), based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced the most contiguous de novo mammalian assembly to date, with chromosome-length scaffolds and only 663 gaps. Our assembly represents a >250-fold improvement in contiguity compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, supporting the most complete repeat family and immune gene complex representation ever produced for a ruminant species.

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.

Therapeutic administration of broadly neutralizing FI6 antibody reveals lack of interaction between human IgG1 and pig Fc receptors

Influenza virus infection is a significant global health threat. Because of the lack of cross-protective universal vaccines, short time window during which antivirals are effective and drug resistance, new therapeutic anti-influenza strategies are required. Broadly, cross-protective antibodies that target conserved sites in the hemagglutinin (HA) stem region have been proposed as therapeutic agents. FI6 is the first proven such monoclonal antibody to bind to H1–H16 and is protective in mice and ferrets. Multiple studies have shown that Fc-dependent mechanisms are essential for FI6 in vivo efficacy. Here, we show that therapeutic administration of FI6 either intravenously or by aerosol to pigs did not reduce viral load in nasal swabs or broncho-alveolar lavage, but aerosol delivery of FI6 reduced gross pathology significantly. We demonstrate that pig Fc receptors do not bind human IgG1 and that FI6 did not mediate antibody-dependent cytotoxicity (ADCC) with pig PBMC, confirming that ADCC is an important mechanism of protection by anti-stem antibodies in vivo. Enhanced respiratory disease, which has been associated with pigs with cross-reactive non-neutralizing anti-HA antibodies, did not occur after FI6 administration. Our results also show that in vitro neutralizing antibody responses are not a robust correlate of protection for the control of influenza infection and pathology in a natural host model.

The major histocompatibility complex homozygous inbred Babraham pig as a resource for veterinary and translational medicine

The Babraham pig is a highly inbred breed first developed in the United Kingdom approximately 50 years ago. Previous reports indicate a very high degree of homozygosity across the genome, including the major histocompatibility complex (MHC) region, but confirmation of homozygosity at the specific MHC loci was lacking. Using both direct sequencing and PCR‐based sequence‐specific typing, we confirm that Babraham pigs are essentially homozygous at their MHC loci and formalise their MHC haplotype as Hp‐55.6. This enhances the utility of the Babraham pig as a useful biomedical model for studies in which controlling for genetic variation is important.

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 (~xa020–30%) compared to that in cattle (~xa05%). 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.

Viral Infection Skews Immunoglobulin Light Chain Repertoire Diversity

Antibody responses are fundamentally important to effector and memory mechanisms of disease resistance. Antibody repertoire diversity and its response to natural infection is poorly understood, yet is a prerequisite for molecular and structural elucidation of functionally protective immunity to viral infections. Using a swine model of mammalian viral infection, we observed marked changes following infection with the major porcine pathogen, porcine reproductive and respiratory syndrome virus (PRRSV). Deep sequencing of >516,000 light chain VJ mRNA genes showed that, similar to humans, swine utilize both lambda and kappa loci equivalently. However, V and J gene usage were highly restricted; ≥99% of lambda light chains were IGLV3 and IGLV8 family members joined to IGLJ2 or IGLJ3, and 100% of kappa locus transcripts were IGKV1 or IGKV2 with only IGKJ2. Complementarity-determining region (CDR) variation was limited. Nevertheless, total diversity richness estimates were 2.3 × 105 for lambda and 1.5 × 105 for kappa, due in part to extensive germline variation in framework regions and allelic variation. Infection by PRRSV reduced total richness due to expression of several highly abundant clonal populations. Antibody light chain repertoires differed substantially among individuals, thus illustrating extensive potential variation in immune response in outbred populations. These findings demonstrate that individual variation in light chain repertoires may be an important component of variable antibody responses to infection and vaccination, and that swine are a relevant model of human antibody diversification in which the immune response capacity is critical to understanding individual variation in immune protection against disease. Conflict of interest statement The authors declare no conflicts of interest. Highlights λ and κ light chain diversity is equivalent to heavy chain diversity High diversity is present despite limited gene segment usage PRRSV infection increases abundance of dominant λ and κ VJ clones High levels of variation are present among animals

The unique evolution of the pig LRC, a single KIR but expansion of LILR and a novel Ig receptor family

The leukocyte receptor complex (LRC) encodes numerous immunoglobulin (Ig)-like receptors involved in innate immunity. These include the killer-cell Ig-like receptors (KIR) and the leukocyte Ig-like receptors (LILR) which can be polymorphic and vary greatly in number between species. Using the recent long-read genome assembly, Sscrofa11.1, we have characterized the porcine LRC on chromosome 6. We identified a ~u2009197-kb region containing numerous LILR genes that were missing in previous assemblies. Out of 17 such LILR genes and fragments, six encode functional proteins, of which three are inhibitory and three are activating, while the majority of pseudogenes had the potential to encode activating receptors. Elsewhere in the LRC, between FCAR and GP6, we identified a novel gene that encodes two Ig-like domains and a long inhibitory intracellular tail. Comparison with two other porcine assemblies revealed a second, nearly identical, non-functional gene encoding a short intracellular tail with ambiguous function. These novel genes were found in a diverse range of mammalian species, including a pseudogene in humans, and typically consist of a single long-tailed receptor and a variable number of short-tailed receptors. Using porcine transcriptome data, both the novel inhibitory gene and the LILR were highly expressed in peripheral blood, while the single KIR gene, KIR2DL1, was either very poorly expressed or not at all. These observations are a prerequisite for improved understanding of immune cell functions in the pig and other species.