April 21, 2020  |  

Comparative analysis of the chicken IFITM locus by targeted genome sequencing reveals evolution of the locus and positive selection in IFITM1 and IFITM3.

The interferon-induced transmembrane (IFITM) protein family comprises a class of restriction factors widely characterised in humans for their potent antiviral activity. Their biological activity is well documented in several animal species, but their genetic variation and biological mechanism is less well understood, particularly in avian species.Here we report the complete sequence of the domestic chicken Gallus gallus IFITM locus from a wide variety of chicken breeds to examine the detailed pattern of genetic variation of the locus on chromosome 5, including the flanking genes ATHL1 and B4GALNT4. We have generated chIFITM sequences from commercial breeds (supermarket-derived chicken breasts), indigenous chickens from Nigeria (Nsukka) and Ethiopia, European breeds and inbred chicken lines from the Pirbright Institute, totalling of 206 chickens. Through mapping of genetic variants to the latest chIFITM consensus sequence our data reveal that the chIFITM locus does not show structural variation in the locus across the populations analysed, despite spanning diverse breeds from different geographic locations. However, single nucleotide variants (SNVs) in functionally important regions of the proteins within certain groups of chickens were detected, in particular the European breeds and indigenous birds from Ethiopia and Nigeria. In addition, we also found that two out of four SNVs located in the chIFITM1 (Ser36 and Arg77) and chIFITM3 (Val103) proteins were simultaneously under positive selection.Together these data suggest that IFITM genetic variation may contribute to the capacities of different chicken populations to resist virus infection.


April 21, 2020  |  

Chromosome-level assembly of the water buffalo genome surpasses human and goat genomes in sequence contiguity.

Rapid innovation in sequencing technologies and improvement in assembly algorithms have enabled the creation of highly contiguous mammalian genomes. Here we report a chromosome-level assembly of the water buffalo (Bubalus bubalis) genome using single-molecule sequencing and chromatin conformation capture data. PacBio Sequel reads, with a mean length of 11.5?kb, helped to resolve repetitive elements and generate sequence contiguity. All five B. bubalis sub-metacentric chromosomes were correctly scaffolded with centromeres spanned. Although the index animal was partly inbred, 58% of the genome was haplotype-phased by FALCON-Unzip. This new reference genome improves the contig N50 of the previous short-read based buffalo assembly more than a thousand-fold and contains only 383 gaps. It surpasses the human and goat references in sequence contiguity and facilitates the annotation of hard to assemble gene clusters such as the major histocompatibility complex (MHC).


October 23, 2019  |  

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M.?mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-?glf strain did not produce the galactofuranose-containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also ‘leaking’ as revealed by a ß-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.


September 22, 2019  |  

Cow, yak, and camel milk diets differentially modulated the systemic immunity and fecal microbiota of rats

Cow milk is most widely consumed; however, non-cattle milk has gained increasing interest because of added nutritive values. We compared the health effects of yak, cow, and camel milk in rats. By measuring several plasma immune factors, significantly more interferon-? was detected in the camel than the yak (P=0.0020) or cow (P=0.0062) milk group. Significantly more IgM was detected in the yak milk than the control group (P=0.0071). The control group had significantly less interleukin 6 than the yak (P=0.0499) and cow (P=0.0248) milk groups. The fecal microbiota of the 144 samples comprised mainly of the Firmicutes (76.70±11.03%), Bacteroidetes (15.27±7.79%), Proteobacteria (3.61±4.34%), and Tenericutes (2.61±2.53%) phyla. Multivariate analyses revealed a mild shift in the fecal microbiota along the milk treatment. We further identified the differential microbes across the four groups. At day 14, 22 and 28 differential genera and species were identified (P=0.0000–0.0462), while 8 and 11 differential genera and species (P=0.0000–0.0013) were found at day 28. Some short-chain fatty acid and succinate producers increased, while certain health-concerned bacteria (Prevotella copri, Phascolarctobacterium faecium, and Bacteroides uniformis) decreased after 14days of yak or camel milk treatment. We demonstrated that different animal milk could confer distinctive nutritive value to the host.


September 22, 2019  |  

Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters.

The present study applied the PacBio single molecule, real-time sequencing technology (SMRT) in evaluating the quality of silage production. Specifically, we produced four types of Medicago sativa silages by using four different lactic acid bacteria-based additives (AD-I, AD-II, AD-III and AD-IV). We monitored the changes in pH, organic acids (including butyric acid, the ratio of acetic acid/lactic acid, ?-aminobutyric acid, 4-hyroxy benzoic acid and phenyl lactic acid), mycotoxins, and bacterial microbiota during silage fermentation. Our results showed that the use of the additives was beneficial to the silage fermentation by enhancing a general pH and mycotoxin reduction, while increasing the organic acids content. By SMRT analysis of the microbial composition in eight silage samples, we found that the bacterial species number and relative abundances shifted apparently after fermentation. Such changes were specific to the LAB species in the additives. Particularly, Bacillus megaterium was the initial dominant species in the raw materials; and after the fermentation process, Pediococcus acidilactici and Lactobacillus plantarum became the most prevalent species, both of which were intrinsically present in the LAB additives. Our data have demonstrated that the SMRT sequencing platform is applicable in assessing the quality of silage.


September 22, 2019  |  

RNA sequencing (RNA-Seq) reveals extremely low levels of reticulocyte-derived globin gene transcripts in peripheral blood from horses (Equus caballus) and cattle (Bos taurus).

RNA-seq has emerged as an important technology for measuring gene expression in peripheral blood samples collected from humans and other vertebrate species. In particular, transcriptomics analyses of whole blood can be used to study immunobiology and develop novel biomarkers of infectious disease. However, an obstacle to these methods in many mammalian species is the presence of reticulocyte-derived globin mRNAs in large quantities, which can complicate RNA-seq library sequencing and impede detection of other mRNA transcripts. A range of supplementary procedures for targeted depletion of globin transcripts have, therefore, been developed to alleviate this problem. Here, we use comparative analyses of RNA-seq data sets generated from human, porcine, equine, and bovine peripheral blood to systematically assess the impact of globin mRNA on routine transcriptome profiling of whole blood in cattle and horses. The results of these analyses demonstrate that total RNA isolated from equine and bovine peripheral blood contains very low levels of globin mRNA transcripts, thereby negating the need for globin depletion and greatly simplifying blood-based transcriptomic studies in these two domestic species.


September 22, 2019  |  

The bacterial microbiome of Dermacentor andersoni ticks influences pathogen susceptibility.

Ticks are of medical importance owing to their ability to transmit pathogens to humans and animals. The Rocky Mountain wood tick, Dermacentor andersoni, is a vector of a number of pathogens, including Anaplasma marginale, which is the most widespread tick-borne pathogen of livestock. Although ticks host pathogenic bacteria, they also harbor bacterial endosymbionts that have a role in tick physiology, survival, as well as pathogen acquisition and transmission. The goal of this study was to characterize the bacterial microbiome and examine the impact of microbiome disruption on pathogen susceptibility. The bacterial microbiome of two populations of D. andersoni with historically different susceptibilities to A. marginale was characterized. In this study, the microbiome was disrupted and then ticks were exposed to A. marginale or Francisella novicida to determine whether the microbiome correlated with pathogen susceptibility. Our study showed that an increase in proportion and quantity of Rickettsia bellii in the microbiome was negatively correlated to A. marginale levels in ticks. Furthermore, a decrease in Francisella endosymbionts was associated with lower F. novicida infection levels, demonstrating a positive pathogen-endosymbiont relationship. We demonstrate that endosymbionts and pathogens have varying interactions, and suggest that microbiome manipulation may provide a possible method for biocontrol by decreasing pathogen susceptibility of ticks.


September 22, 2019  |  

Analysis of the duodenal microbiotas of weaned piglet fed with epidermal growth factor-expressed Saccharomyces cerevisiae.

The bacterial community of the small intestine is a key factor that has strong influence on the health of gastrointestinal tract (GIT) in mammals during and shortly after weaning. The aim of this study was to analyze the effects of the diets of supplemented with epidermal growth factor (EGF)-expressed Saccharomyces cerevisiae (S. cerevisiae) on the duodenal microbiotas of weaned piglets.Revealed in this study, at day 7, 14 and 21, respectively, the compositional sequencing analysis of the 16S rRNA in the duodenum had no marked difference in microbial diversity from the phylum to species levels between the INVSc1(EV) and other recombinant strains encompassing INVSc1-EE(+), INVSc1-TE(-), and INVSc1-IE(+). Furthermore, the populations of potentially enterobacteria (e.g., Clostridium and Prevotella) and probiotic (e.g., Lactobacilli and Lactococcus) also remained unchanged among recombinant S. cerevisiae groups (P?>?0.05). However, the compositional sequencing analysis of the 16S rRNA in the duodenum revealed significant difference in microbial diversity from phylum to species levels between the control group and recombinant S. cerevisiae groups. In terms of the control group (the lack of S. cerevisiae), these data confirmed that dietary exogenous S. cerevisiae had the feasibility to be used as a supplement for enhancing potentially probiotic (e.g., Lactobacilli and Lactococcus) (P?


September 22, 2019  |  

Effects of metal and metalloid pollutants on the microbiota composition of feces obtained from twelve commercial pig farms across China.

Understanding the metal and metalloid contamination and microbiota composition of pig feces is an important step required to support the design and implementation of effective pollution control and prevention strategies. A survey was implemented in 12 locations across China to investigate the content of metals and metalloids, and the main composition of the microbial communities of commercially reared pigs during two growth periods, defined as the early (Q group) and the later fattening growth phases (H group). These data showed widespread Al, Mn, Cu, Zn, and Fe pollution in pig feces. The concentration of Zn in the Q group feces was nearly two times higher than the levels measured in the H group. The microbial composition of the Q group exhibited greater richness of operational taxonomic units (OTUs) and fewer bacteria associated with zoonotic diseases compared with the microbial composition of the H group. Spearman rank correlation analysis showed that Cu and northern latitudes had a significant positive effect on the richness of bacterial communities in pig feces. Zn and Cd exhibited the biggest impact on microbial community composition based on canonical correspondence analysis. Functional metagenomic prediction indicated that about 0.8% genes present in the pig feces bacteria community are related to human diseases, and significantly more predicted pathogenic genes were detected in the H group than in the Q group. These results support the need to monitor heavy metal contamination and to control for zoonotic pathogens disseminated from pig feces in Chinese pig farms. Copyright © 2018. Published by Elsevier B.V.


September 22, 2019  |  

A high-quality annotated transcriptome of swine peripheral blood.

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.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.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.


September 22, 2019  |  

A survey of transcriptome complexity in Sus scrofa using single-molecule long-read sequencing.

Alternative splicing (AS) and fusion transcripts produce a vast expansion of transcriptomes and proteomes diversity. However, the reliability of these events and the extend of epigenetic mechanisms have not been adequately addressed due to its limitation of uncertainties about the complete structure of mRNA. Here we combined single-molecule real-time sequencing, Illumina RNA-seq and DNA methylation data to characterize the landscapes of DNA methylation on AS, fusion isoforms formation and lncRNA feature and further to unveil the transcriptome complexity of pig. Our analysis identified an unprecedented scale of high-quality full-length isoforms with over 28,127 novel isoforms from 26,881 novel genes. More than 92,000 novel AS events were detected and intron retention predominated in AS model, followed by exon skipping. Interestingly, we found that DNA methylation played an important role in generating various AS isoforms by regulating splicing sites, promoter regions and first exons. Furthermore, we identified a large of fusion transcripts and novel lncRNAs, and found that DNA methylation of the promoter and gene body could regulate lncRNA expression. Our results significantly improved existed gene models of pig and unveiled that pig AS and epigenetic modify were more complex than previously thought.


September 22, 2019  |  

Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human.

Despite the significance of chicken as a model organism, our understanding of the chicken transcriptome is limited compared to human. This issue is common to all non-human vertebrate annotations due to the difficulty in transcript identification from short read RNAseq data. While previous studies have used single molecule long read sequencing for transcript discovery, they did not perform RNA normalization and 5′-cap selection which may have resulted in lower transcriptome coverage and truncated transcript sequences.We sequenced normalised chicken brain and embryo RNA libraries with Pacific Bioscience Iso-Seq. 5′ cap selection was performed on the embryo library to provide methodological comparison. From these Iso-Seq sequencing projects, we have identified 60 k transcripts and 29 k genes within the chicken transcriptome. Of these, more than 20 k are novel lncRNA transcripts with ~3 k classified as sense exonic overlapping lncRNA, which is a class that is underrepresented in many vertebrate annotations. The relative proportion of alternative transcription events revealed striking similarities between the chicken and human transcriptomes while also providing explanations for previously observed genomic differences.Our results indicate that the chicken transcriptome is similar in complexity compared to human, and provide insights into other vertebrate biology. Our methodology demonstrates the potential of Iso-Seq sequencing to rapidly expand our knowledge of transcriptomics.


September 22, 2019  |  

Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection.

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ~75% of the genus-level bacterial and archaeal taxa present in the rumen.


September 22, 2019  |  

Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire.

Vertebrate immune systems generate diverse repertoires of antibodies capable of mediating response to a variety of antigens. Next generation sequencing methods provide unique approaches to a number of immuno-based research areas including antibody discovery and engineering, disease surveillance, and host immune response to vaccines. In particular, single-molecule circular consensus sequencing permits the sequencing of antibody repertoires at previously unattainable depths of coverage and accuracy. We approached the bovine immunoglobulin G (IgG) repertoire with the objective of characterizing diversity of expressed IgG transcripts. Here we present single-molecule real-time sequencing data of expressed IgG heavy-chain repertoires of four individual cattle. We describe the diversity observed within antigen binding regions and visualize this diversity using a network-based approach.We generated 49,945 high quality cDNA sequences, each spanning the entire IgG variable region from four Bos taurus calves. From these sequences we identified 49,521 antigen binding regions using the automated Paratome web server. Approximately 9% of all unique complementarity determining 2 (CDR2) sequences were of variable lengths. A bimodal distribution of unique CDR3 sequence lengths was observed, with common lengths of 5-6 and 21-25 amino acids. The average number of cysteine residues in CDR3s increased with CDR3 length and we observed that cysteine residues were centrally located in CDR3s. We identified 19 extremely long CDR3 sequences (up to 62 amino acids in length) within IgG transcripts. Network analyses revealed distinct patterns among the expressed IgG antigen binding repertoires of the examined individuals.We utilized circular consensus sequencing technology to provide baseline data of the expressed bovine IgG repertoire that can be used for future studies important to livestock research. Somatic mutation resulting in base insertions and deletions in CDR2 further diversifies the bovine antibody repertoire. In contrast to previous studies, our data indicate that unusually long CDR3 sequences are not unique to IgM antibodies in cattle. Centrally located cysteine residues in bovine CDR3s provide further evidence that disulfide bond formation is likely of structural importance. We hypothesize that network or cluster-based analyses of expressed antibody repertoires from controlled challenge experiments will help identify novel natural antigen binding solutions to specific pathogens of interest.


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