High-volume hybridoma sequencing on the NeuroMabSeq platform enables efficient generation of recombinant monoclonal antibodies and scFvs for neuroscience research.

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

NeuroMabSeq: high volume acquisition, processing, and curation of hybridoma sequences and their use in generating recombinant monoclonal antibodies and scFvs for neuroscience research.

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as searchable DNA sequence database ( neuromabseq.ucdavis.edu ) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

A two-stage digestion of whole murine knee joints for single-cell RNA sequencing.

Objective: Single-cell RNA sequencing (scRNA-seq) is a powerful technology that can be applied to the cells populating the whole knee in the study of joint pathology. The knee contains cells embedded in hard structural tissues, cells in softer tissues and membranes, and immune cells. This creates a technical challenge in preparing a viable and representative cell suspension suitable for use in scRNA-seq in minimal time, where under-digestion may exclude cells in hard tissues, over-digestion may damage soft tissue cells, and prolonged digestion may induce phenotypic drift. We developed a rapid two-stage digestion protocol to overcome these difficulties. Design: A two-stage digest consisting of first collagenase IV, an intermediate cell recovery, then collagenase II on the remaining hard tissue. Cells were sequenced on the 10x Genomics platform. Results: We observed consistent cell numbers and viable single cell suspensions suitable for scRNA-seq analysis. Comparison of contralateral knees and separate mice showed reproducible cell yields and gene expression patterns by similar cell-types. A diverse collection of structural and immune cells were captured with a majority from immune origins. Two digestions were necessary to capture all cell-types. Conclusions: The knee contains a diverse mixture of stromal and immune cells that may be crucial for the study of osteoarthritis. The two-stage digestion presented here reproducibly generated highly viable and representative single-cell suspension for sequencing from the whole knee. This protocol facilitates transcriptomic studies of the joint as a complete organ.

Draft genome assemblies using sequencing reads from Oxford Nanopore Technology and Illumina platforms for four species of North American Fundulus killifish.

BACKGROUND: Whole-genome sequencing data from wild-caught individuals of closely related North American killifish species (Fundulus xenicus, Fundulus catenatus, Fundulus nottii, and Fundulus olivaceus) were obtained using long-read Oxford Nanopore Technology (ONT) PromethION and short-read Illumina platforms. FINDINGS: Draft de novo reference genome assemblies were generated using a combination of long and short sequencing reads. For each species, the PromethION platform was used to generate 30-45× sequence coverage, and the Illumina platform was used to generate 50-160× sequence coverage. Illumina-only assemblies were fragmented with high numbers of contigs, while ONT-only assemblies were error prone with low BUSCO scores. The highest N50 values, ranging from 0.4 to 2.7 Mb, were from assemblies generated using a combination of short- and long-read data. BUSCO scores were consistently >90% complete using the Eukaryota database. CONCLUSIONS: High-quality genomes can be obtained from a combination of using short-read Illumina data to polish assemblies generated with long-read ONT data. Draft assemblies and raw sequencing data are available for public use. We encourage use and reuse of these data for assembly benchmarking and other analyses.

The genome sequence of segmental allotetraploid peanut Arachis hypogaea.

Like many other crops, the cultivated peanut (Arachis hypogaea L.) is of hybrid origin and has a polyploid genome that contains essentially complete sets of chromosomes from two ancestral species. Here we report the genome sequence of peanut and show that after its polyploid origin, the genome has evolved through mobile-element activity, deletions and by the flow of genetic information between corresponding ancestral chromosomes (that is, homeologous recombination). Uniformity of patterns of homeologous recombination at the ends of chromosomes favors a single origin for cultivated peanut and its wild counterpart A. monticola. However, through much of the genome, homeologous recombination has created diversity. Using new polyploid hybrids made from the ancestral species, we show how this can generate phenotypic changes such as spontaneous changes in the color of the flowers. We suggest that diversity generated by these genetic mechanisms helped to favor the domestication of the polyploid A. hypogaea over other diploid Arachis species cultivated by humans.

Transcriptome Analysis of Salmonella Heidelberg after Exposure to Cetylpyridinium Chloride, Acidified Calcium Hypochlorite, and Peroxyacetic Acid.

The application of RNA sequencing in commercial poultry could facilitate a novel approach toward food safety with respect to identifying conditions in food production that mitigate transcription of genes associated with virulence and survivability. In this study, we evaluated the effects of disinfectant exposure on the transcriptomes of two field isolates of Salmonella Heidelberg (SH) isolated from a commercial broiler processing plant in 1992 and 2014. The isolates were each exposed separately to the following disinfectants commonly used in poultry processing: cetylpyridinium chloride (CPC), acidified calcium hypochlorite (aCH), and peroxyacetic acid (PAA). Exposure times were 8 s with CPC to simulate a poultry processing dipping station or 90 min with aCH and PAA to simulate the chiller tank in a poultry processing plant at 4°C. Based on comparison with a publicly available annotated SH reference genome with 5,088 genes, 90 genes were identified as associated with virulence, pathogenicity, and resistance (VPR). Of these 90 VPR genes, 9 (10.0%), 28 (31.1%), and 1 (1.1%) gene were upregulated in SH 2014 and 21 (23.3%), 26 (28.9%), and 2 (2.2%) genes were upregulated in SH 2014 challenged with CPC, aCH, and PAA, respectively. This information and previously reported MICs for the three disinfectants with both SH isolates allow researchers to make more accurate recommendations regarding control methods of SH and public health considerations related to SH in food production facilities where SH has been isolated. For example, the MICs revealed that aCH is ineffective for SH inhibition at regulatory levels allowed for poultry processing and that aCH was ineffective for inhibiting SH growth and caused an upregulation of VPR genes.

Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce.

Lettuce (Lactuca sativa) is a major crop and a member of the large, highly successful Compositae family of flowering plants. Here we present a reference assembly for the species and family. This was generated using whole-genome shotgun Illumina reads plus in vitro proximity ligation data to create large superscaffolds; it was validated genetically and superscaffolds were oriented in genetic bins ordered along nine chromosomal pseudomolecules. We identify several genomic features that may have contributed to the success of the family, including genes encoding Cycloidea-like transcription factors, kinases, enzymes involved in rubber biosynthesis and disease resistance proteins that are expanded in the genome. We characterize 21 novel microRNAs, one of which may trigger phasiRNAs from numerous kinase transcripts. We provide evidence for a whole-genome triplication event specific but basal to the Compositae. We detect 26% of the genome in triplicated regions containing 30% of all genes that are enriched for regulatory sequences and depleted for genes involved in defence.

The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut.

Cultivated peanut (Arachis hypogaea) is an allotetraploid with closely related subgenomes of a total size of ∼2.7 Gb. This makes the assembly of chromosomal pseudomolecules very challenging. As a foundation to understanding the genome of cultivated peanut, we report the genome sequences of its diploid ancestors (Arachis duranensis and Arachis ipaensis). We show that these genomes are similar to cultivated peanut's A and B subgenomes and use them to identify candidate disease resistance genes, to guide tetraploid transcript assemblies and to detect genetic exchange between cultivated peanut's subgenomes. On the basis of remarkably high DNA identity of the A. ipaensis genome and the B subgenome of cultivated peanut and biogeographic evidence, we conclude that A. ipaensis may be a direct descendant of the same population that contributed the B subgenome to cultivated peanut.

The genome sequence of the outbreeding globe artichoke constructed de novo incorporating a phase-aware low-pass sequencing strategy of F1 progeny.

Globe artichoke (Cynara cardunculus var. scolymus) is an out-crossing, perennial, multi-use crop species that is grown worldwide and belongs to the Compositae, one of the most successful Angiosperm families. We describe the first genome sequence of globe artichoke. The assembly, comprising of 13,588 scaffolds covering 725 of the 1,084 Mb genome, was generated using ~133-fold Illumina sequencing data and encodes 26,889 predicted genes. Re-sequencing (30×) of globe artichoke and cultivated cardoon (C. cardunculus var. altilis) parental genotypes and low-coverage (0.5 to 1×) genotyping-by-sequencing of 163 F1 individuals resulted in 73% of the assembled genome being anchored in 2,178 genetic bins ordered along 17 chromosomal pseudomolecules. This was achieved using a novel pipeline, SOILoCo (Scaffold Ordering by Imputation with Low Coverage), to detect heterozygous regions and assign parental haplotypes with low sequencing read depth and of unknown phase. SOILoCo provides a powerful tool for de novo genome analysis of outcrossing species. Our data will enable genome-scale analyses of evolutionary processes among crops, weeds, and wild species within and beyond the Compositae, and will facilitate the identification of economically important genes from related species.

Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms.

CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform-specific antibodies. The canine (Canis familiaris) CD1 locus was previously found to contain three functional CD1A genes: canCD1A2, canCD1A6, and canCD1A8, where two variants of canCD1A8, canCD1A8.1 and canCD1A8.2, were assumed to be allelic variants. However, we hypothesized that these rather represented two separate genes. Sequencing of three overlapping bacterial artificial chromosomes (BACs) spanning the entire canine CD1 locus revealed canCD1A8.2 and canCD1A8.1 to be located in tandem between canCD1A7 and canCD1C, and canCD1A8.1 was consequently renamed canCD1A9. Green fluorescent protein (GFP)-fused canine CD1 transcripts were recombinantly expressed in 293T cells. All proteins showed a highly positive GFP expression except for canine CD1d and a splice variant of canine CD1a8 lacking exon 3. Probing with a panel of anti-CD1 monoclonal antibodies (mAbs) showed that Ca13.9H11 and Ca9.AG5 only recognized canine CD1a8 and CD1a9 isoforms, and Fe1.5F4 mAb solely recognized canine CD1a6. Anti-CD1b mAbs recognized the canine CD1b protein, but also bound CD1a2, CD1a8, and CD1a9. Interestingly, Ca9.AG5 showed allele specificity based on a single nucleotide polymorphism (SNP) located at position 321. Our findings have refined the structure of the canine CD1 locus and available antibody specificity against canine CD1 proteins. These are important fundamentals for future investigation of the role of canine CD1 in lipid immunity.

Genome Sequence and Architecture of the Tobacco Downy Mildew Pathogen Peronospora tabacina.

Peronospora tabacina is an obligate biotrophic oomycete that causes blue mold or downy mildew on tobacco (Nicotiana tabacum). It is an economically important disease occurring frequently in tobacco-growing regions worldwide. We sequenced and characterized the genomes of two P. tabacina isolates and mined them for pathogenicity-related proteins and effector-encoding genes. De novo assembly of the genomes using Illumina reads resulted in 4,016 (63.1 Mb, N50 = 79 kb) and 3,245 (55.3 Mb, N50 = 61 kb) scaffolds for isolates 968-J2 and 968-S26, respectively, with an estimated genome size of 68 Mb. The mitochondrial genome has a similar size (approximately 43 kb) and structure to those of other oomycetes, plus several minor unique features. Repetitive elements, primarily retrotransposons, make up approximately 24% of the nuclear genome. Approximately 18,000 protein-coding gene models were predicted. Mining the secretome revealed approximately 120 candidate RxLR, six CRN (candidate effectors that elicit crinkling and necrosis), and 61 WY domain-containing proteins. Candidate RxLR effectors were shown to be predominantly undergoing diversifying selection, with approximately 57% located in variable gene-sparse regions of the genome. Aligning the P. tabacina genome to Hyaloperonospora arabidopsidis and Phytophthora spp. revealed a high level of synteny. Blocks of synteny show gene inversions and instances of expansion in intergenic regions. Extensive rearrangements of the gene-rich genomic regions do not appear to have occurred during the evolution of these highly variable pathogens. These assemblies provide the basis for studies of virulence in this and other downy mildew pathogens.

Strain- and plasmid-level deconvolution of a synthetic metagenome by sequencing proximity ligation products.

Metagenomics is a valuable tool for the study of microbial communities but has been limited by the difficulty of "binning" the resulting sequences into groups corresponding to the individual species and strains that constitute the community. Moreover, there are presently no methods to track the flow of mobile DNA elements such as plasmids through communities or to determine which of these are co-localized within the same cell. We address these limitations by applying Hi-C, a technology originally designed for the study of three-dimensional genome structure in eukaryotes, to measure the cellular co-localization of DNA sequences. We leveraged Hi-C data generated from a simple synthetic metagenome sample to accurately cluster metagenome assembly contigs into groups that contain nearly complete genomes of each species. The Hi-C data also reliably associated plasmids with the chromosomes of their host and with each other. We further demonstrated that Hi-C data provides a long-range signal of strain-specific genotypes, indicating such data may be useful for high-resolution genotyping of microbial populations. Our work demonstrates that Hi-C sequencing data provide valuable information for metagenome analyses that are not currently obtainable by other methods. This metagenomic Hi-C method could facilitate future studies of the fine-scale population structure of microbes, as well as studies of how antibiotic resistance plasmids (or other genetic elements) mobilize in microbial communities. The method is not limited to microbiology; the genetic architecture of other heterogeneous populations of cells could also be studied with this technique.

RNAseq-based transcriptome analysis of Lactuca sativa infected by the fungal necrotroph Botrytis cinerea.

The fungal pathogen Botrytis cinerea establishes a necrotrophic interaction with its host plants, including lettuce (Lactuca sativa), causing it to wilt, collapse and eventually dry up and die, which results in serious economic losses. Global expression profiling using RNAseq and the newly sequenced lettuce genome identified a complex network of genes involved in the lettuce-B. cinerea interaction. The observed high number of differentially expressed genes allowed us to classify them according to the biological pathways in which they are implicated, generating a holistic picture. Most pronounced were the induction of the phenylpropanoid pathway and terpenoid biosynthesis, whereas photosynthesis was globally down-regulated at 48 h post-inoculation. Large-scale comparison with data available on the interaction of B. cinerea with the model plant Arabidopsis thaliana revealed both general and species-specific responses to infection with this pathogen. Surprisingly, expression analysis of selected genes could not detect significant systemic transcriptional alterations in lettuce leaves distant from the inoculation site. Additionally, we assessed the response of these lettuce genes to a biotrophic pathogen, Bremia lactucae, revealing that similar pathways are induced during compatible interactions of lettuce with necrotrophic and biotrophic pathogens.

Consequences of normalizing transcriptomic and genomic libraries of plant genomes using a duplex-specific nuclease and tetramethylammonium chloride.

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce.

Enhancing radiation hybrid mapping through whole genome amplification.

Radiation hybrid (RH) mapping is limited by the inherent genomic instability of RH clones entailing both, limited DNA sample amounts and genomic heterogeneity of the clones. Here the instability of RH clones is quantified and the suitability of the multiple strand displacement whole genome amplification method (WGA) for radiation hybrid mapping is assessed. To quantify the instability of RH clones, eleven clones of a 10,000(Rad) rhesus macaque radiation hybrid panel were passaged ten times and analyzed by interspersed repeat sequence specific quantitative PCR and by genotyping of 46 macaque chromosome 5 STS markers. The quantitative PCR data indicate an average loss of 55% of the donor DNA over 10 passages. Over the same period, a dropout of 46.2% of the STS markers was observed. These data indicate a genome wide half-life of the donor DNA of 8.7 passages and of 10.6 passages for the chromosome 5 markers. The genotyping data of the genomic RH DNA were compared to three sets of WGA experiments: 1) single wgaDNA amplifications, 2) six WGA replicates, and 3) re-amplification of wga DNA. The assays demonstrated concordance rates of 97.6%, 98% and 99.3%, respectively, and indicated the marker specificity of some repeated WGA dropouts. The study confirms that WGA is suitable for RH mapping studies should enable the accurate analysis of almost an infinite numbers of markers. WGA will allow the analysis of earliest RH clone passages, thus limiting their heterogeneity and RH mapping artifacts.

Gene synteny comparisons between different vertebrates provide new insights into breakage and fusion events during mammalian karyotype evolution.

BACKGROUND: Genome comparisons have made possible the reconstruction of the eutherian ancestral karyotype but also have the potential to provide new insights into the evolutionary inter-relationship of the different eutherian orders within the mammalian phylogenetic tree. Such comparisons can additionally reveal (i) the nature of the DNA sequences present within the evolutionary breakpoint regions and (ii) whether or not the evolutionary breakpoints occur randomly across the genome. Gene synteny analysis (E-painting) not only greatly reduces the complexity of comparative genome sequence analysis but also extends its evolutionary reach. RESULTS: E-painting was used to compare the genome sequences of six different mammalian species and chicken. A total of 526 evolutionary breakpoint intervals were identified and these were mapped to a median resolution of 120 kb, the highest level of resolution so far obtained. A marked correlation was noted between evolutionary breakpoint frequency and gene density. This correlation was significant not only at the chromosomal level but also sub-chromosomally when comparing genome intervals of lengths as short as 40 kb. Contrary to previous findings, a comparison of evolutionary breakpoint locations with the chromosomal positions of well mapped common fragile sites and cancer-associated breakpoints failed to reveal any evidence for significant co-location. Primate-specific chromosomal rearrangements were however found to occur preferentially in regions containing segmental duplications and copy number variants. CONCLUSION: Specific chromosomal regions appear to be prone to recurring rearrangement in different mammalian lineages ('breakpoint reuse') even if the breakpoints themselves are likely to be non-identical. The putative ancestral eutherian genome, reconstructed on the basis of the synteny analysis of 7 vertebrate genome sequences, not only confirmed the results of previous molecular cytogenetic studies but also increased the definition of the inferred structure of ancestral eutherian chromosomes. For the first time in such an analysis, the opossum was included as an outgroup species. This served to confirm our previous model of the ancestral eutherian genome since all ancestral syntenic segment associations were also noted in this marsupial.

Changes of higher order chromatin arrangements during major genome activation in bovine preimplantation embryos.

Gene-dense chromosome territories (CTs) are typically located more interior, gene-poor CTs more peripheral in mammalian cell nuclei. Here, we show that this gene-density correlated CT positioning holds for the most gene-rich and gene-poor bovine chromosomes 19 and 20, respectively, in bovine fibroblast and lymphocyte nuclei. In order to determine the period at which this non-random CT order is established during development, we performed fluorescence in situ hybridization, on three-dimensionally preserved bovine preimplantation embryos generated by in vitro fertilization and investigated the distribution of BTA 19 and 20 CTs. Radial arrangements of CTs 19 and 20 were the same up to the 8-cell stage. At the 10- to 16-cell stage, however, a significant difference became apparent with CTs 19 localized more internally and CTs 20 more peripherally. Since major genome activation in bovine embryos occurs at the 8- to 16-cell stage, our findings demonstrate a temporal correlation between transcriptional activation and a major rearrangement of chromatin topography in blastomere nuclei.

Effects of environmental tobacco smoke in vivo on rhesus monkey semen quality, sperm function, and sperm metabolism.

The objective of this study was to use a non-human primate model to examine the effect of environmental tobacco smoke (ETS) in vivo on semen quality, sperm function, and sperm metabolism. Four adult rhesus macaques (Macaca mulatta) were exposed to ETS for six months, and semen samples were collected every week for evaluation. ETS exposure in vivo did not affect semen quality and sperm function. The sperm X:Y chromosome ratio remained unchanged after ETS exposure. The sex ratio of the embryos fertilized by ETS-exposed males was not different from the control male. However, sperm showed changes in metabolome detected by NMR during the ETS exposure. We concluded that with the duration and level of ETS exposure in this study, semen quality and sperm function were not affected, whereas sperm did undergo metabolic changes with ETS exposure in vivo.

Nothing 'FISH'y about the rhesus macaque sex ratio.

BACKGROUND: The rhesus macaque is an important model to study the effects of environmental toxicants on human reproduction. While the offspring sex ratio is one of the measurements in reproductive studies, this ratio has not been established for rhesus macaques. METHODS: The birth data for the last 23 years at the California National Primate Research Center are reported to determine the post-zygotic sex ratio. The percentage of X- and Y-bearing sperm was evaluated in four males by fluorescence in situ hybridization and by quantitative real-time polymerase chain reaction (QRT-PCR) to determine the pre-zygotic sex ratio. RESULTS: The total birth data showed a male-to-female offspring sex ratio of 1.03, and the observed ratio of Y- and X-bearing spermatozoa was 1.01. The QRT-PCR failed to provide precise and consistent results. CONCLUSIONS: The offspring sex ratio and sperm X:Y ratio data provide a reference for future studies of the reproductive toxicology in rhesus macaques.