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September 22, 2019  |  

Next-generation approaches to advancing eco-immunogenomic research in critically endangered primates.

High-throughput sequencing platforms are generating massive amounts of genomic data from nonmodel species, and these data sets are valuable resources that can be mined to advance a number of research areas. An example is the growing amount of transcriptome data that allow for examination of gene expression in nonmodel species. Here, we show how publicly available transcriptome data from nonmodel primates can be used to design novel research focused on immunogenomics. We mined transcriptome data from the world’s most endangered group of primates, the lemurs of Madagascar, for sequences corresponding to immunoglobulins. Our results confirmed homology between strepsirrhine and haplorrhine primate immunoglobulins and allowed for high-throughput sequencing of expressed antibodies (Ig-seq) in Coquerel’s sifaka (Propithecus coquereli). Using both Pacific Biosciences RS and Ion Torrent PGM sequencing, we performed Ig-seq on two individuals of Coquerel’s sifaka. We generated over 150 000 sequences of expressed antibodies, allowing for molecular characterization of the antigen-binding region. Our analyses suggest that similar VDJ expression patterns exist across all primates, with sequences closely related to the human VH 3 immunoglobulin family being heavily represented in sifaka antibodies. Moreover, the antigen-binding region of sifaka antibodies exhibited similar amino acid variation with respect to haplorrhine primates. Our study represents the first attempt to characterize sequence diversity of the expressed antibody repertoire in a species of lemur. We anticipate that methods similar to ours will provide the framework for investigating the adaptive immune response in wild populations of other nonmodel organisms and can be used to advance the burgeoning field of eco-immunology. © 2014 John Wiley & Sons Ltd.


September 22, 2019  |  

Comparative Annotation Toolkit (CAT)-simultaneous clade and personal genome annotation.

The recent introductions of low-cost, long-read, and read-cloud sequencing technologies coupled with intense efforts to develop efficient algorithms have made affordable, high-quality de novo sequence assembly a realistic proposition. The result is an explosion of new, ultracontiguous genome assemblies. To compare these genomes, we need robust methods for genome annotation. We describe the fully open source Comparative Annotation Toolkit (CAT), which provides a flexible way to simultaneously annotate entire clades and identify orthology relationships. We show that CAT can be used to improve annotations on the rat genome, annotate the great apes, annotate a diverse set of mammals, and annotate personal, diploid human genomes. We demonstrate the resulting discovery of novel genes, isoforms, and structural variants-even in genomes as well studied as rat and the great apes-and how these annotations improve cross-species RNA expression experiments.© 2018 Fiddes et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Avian transcriptomics: opportunities and challenges

Recent developments in next-generation sequencing technologies have greatly facilitated the study of whole transcriptomes in model and non-model species. Studying the transcriptome and how it changes across a variety of biological conditions has had major implications for our understanding of how the genome is regulated in different contexts, and how to interpret adaptations and the phenotype of an organism. The aim of this review is to highlight the potential of these new technologies for the study of avian transcriptomics, and to summarise how transcriptomics has been applied in ornithology. A total of 81 peer-reviewed scientific articles that used transcriptomics to answer questions within a broad range of study areas in birds are used as examples throughout the review. We further provide a quick guide to highlight the most important points which need to be take into account when planning a transcriptomic study in birds, and discuss how researchers with little background in molecular biology can avoid potential pitfalls. Suggestions for further reading are supplied throughout. We also discuss possible future developments in the technology platforms used for ribonucleic acid sequencing. By summarising how these novel technologies can be used to answer questions that have long been asked by ornithologists, we hope to bridge the gap between traditional ornithology and genomics, and to stimulate more interdisciplinary research.


September 22, 2019  |  

Full-length transcriptome of Misgurnus anguillicaudatus provides insights into evolution of genus Misgurnus.

Reconstruction and annotation of transcripts, particularly for a species without reference genome, plays a critical role in gene discovery, investigation of genomic signatures, and genome annotation in the pre-genomic era. This study generated 33,330 full-length transcripts of diploid M. anguillicaudatus using PacBio SMRT Sequencing. A total of 6,918 gene families were identified with two or more isoforms, and 26,683 complete ORFs with an average length of 1,497?bp were detected. Totally, 1,208 high-confidence lncRNAs were identified, and most of these appeared to be precursor transcripts of miRNAs or snoRNAs. Phylogenetic tree of the Misgurnus species was inferred based on the 1,905 single copy orthologous genes. The tetraploid and diploid M. anguillicaudatus grouped into a clade, and M. bipartitus showed a closer relationship with the M. anguillicaudatus. The overall evolutionary rates of tetraploid M. anguillicaudatus were significantly higher than those of other Misgurnus species. Meanwhile, 28 positively selected genes were identified in M. anguillicaudatus clade. These positively selected genes may play critical roles in the adaptation to various habitat environments for M. anguillicaudatus. This study could facilitate further exploration of the genomic signatures of M. anguillicaudatus and provide potential insights into unveiling the evolutionary history of tetraploid loach.


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.


September 22, 2019  |  

First insights into the nature and evolution of antisense transcription in nematodes.

The development of multicellular organisms is coordinated by various gene regulatory mechanisms that ensure correct spatio-temporal patterns of gene expression. Recently, the role of antisense transcription in gene regulation has moved into focus of research. To characterize genome-wide patterns of antisense transcription and to study their evolutionary conservation, we sequenced a strand-specific RNA-seq library of the nematode Pristionchus pacificus.We identified 1112 antisense configurations of which the largest group represents 465 antisense transcripts (ASTs) that are fully embedded in introns of their host genes. We find that most ASTs show homology to protein-coding genes and are overrepresented in proteomic data. Together with the finding, that expression levels of ASTs and host genes are uncorrelated, this indicates that most ASTs in P. pacificus do not represent non-coding RNAs and do not exhibit regulatory functions on their host genes. We studied the evolution of antisense gene pairs across 20 nematode genomes, showing that the majority of pairs is lineage-specific and even the highly conserved vps-4, ddx-27, and sel-2 loci show abundant structural changes including duplications, deletions, intron gains and loss of antisense transcription. In contrast, host genes in general, are remarkably conserved and encode exceptionally long introns leading to unusually large blocks of conserved synteny.Our study has shown that in P. pacificus antisense transcription as such does not define non-coding RNAs but is rather a feature of highly conserved genes with long introns. We hypothesize that the presence of regulatory elements imposes evolutionary constraint on the intron length, but simultaneously, their large size makes them a likely target for translocation of genomic elements including protein-coding genes that eventually end up as ASTs.


September 22, 2019  |  

Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.


September 22, 2019  |  

Alternative isoform analysis of Ttc8 expression in the rat pineal gland using a multi-platform sequencing approach reveals neural regulation.

Alternative isoform regulation (AIR) vastly increases transcriptome diversity and plays an important role in numerous biological processes and pathologies. However, the detection and analysis of isoform-level differential regulation is difficult, particularly in the face of complex and incompletely-annotated transcriptomes. Here we have used Illumina short-read/high-throughput RNA-Seq to identify 55 genes that exhibit neurally-regulated AIR in the pineal gland, and then used two other complementary experimental platforms to further study and characterize the Ttc8 gene, which is involved in Bardet-Biedl syndrome and non-syndromic retinitis pigmentosa. Use of the JunctionSeq analysis tool led to the detection of several novel exons and splice junctions in this gene, including two novel alternative transcription start sites which were found to display disproportionately strong neurally-regulated differential expression in several independent experiments. These high-throughput sequencing results were validated and augmented via targeted qPCR and long-read Pacific Biosciences SMRT sequencing. We confirmed the existence of numerous novel splice junctions and the selective upregulation of the two novel start sites. In addition, we identified more than 20 novel isoforms of the Ttc8 gene that are co-expressed in this tissue. By using information from multiple independent platforms we not only greatly reduce the risk of errors, biases, and artifacts influencing our results, we also are able to characterize the regulation and splicing of the Ttc8 gene more deeply and more precisely than would be possible via any single platform. The hybrid method outlined here represents a powerful strategy in the study of the transcriptome.


September 22, 2019  |  

Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.

Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1a, Nrxn1ß, Nrxn2ß, Nrxn3a, and Nrxn3ß mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1a and Nrxn3a (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-a, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that a-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.


September 22, 2019  |  

A manganese superoxide dismutase (MnSOD) from red lip mullet, Liza haematocheila: Evaluation of molecular structure, immune response, and antioxidant function.

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant metalloenzyme. The main function of this enzyme is to dismutase the toxic superoxide anion (O2-) into less toxic hydrogen peroxide (H2O2) and oxygen (O2). Structural analysis of mullet MnSOD (MuMnSOD) was performed using different bioinformatics tools. Pairwise alignment revealed that the protein sequence matched to that derived from Larimichthys crocea with a 95.2% sequence identity. Phylogenetic tree analysis showed that the MuMnSOD was included in the category of teleosts. Multiple sequence alignment showed that a SOD Fe-N domain, SOD Fe-C domain, and Mn/Fe SOD signature were highly conserved among the other examined MnSOD orthologs. Quantitative real-time PCR showed that the highest MuMnSOD mRNA expression level was in blood cells. The highest expression level of MuMnSOD was observed in response to treatment with both Lactococcus garvieae and lipopolysaccharide (LPS) at 6?h post treatment in the head kidney and blood. Potential ROS-scavenging ability of the purified recombinant protein (rMuMnSOD) was examined by the xanthine oxidase assay (XOD assay). The optimum temperature and pH for XOD activity were found to be 25?°C and pH 7, respectively. Relative XOD activity was significantly increased with the dose of rMuMnSOD, revealing its dose dependency. Activity of rMuMnSOD was inhibited by potassium cyanide (KCN) and N-N’-diethyl-dithiocarbamate (DDC). Moreover, expression of MuMnSOD resulted in considerable growth retardation of both gram-positive and gram-negative bacteria. Results of the current study suggest that MuMnSOD acts as an antioxidant enzyme and participates in the immune response in mullet. Copyright © 2018 Elsevier Ltd. All rights reserved.


September 22, 2019  |  

Long non-coding RNA identification: comparing machine learning based tools for long non-coding transcripts discrimination

Long noncoding RNA (lncRNA) is a kind of noncoding RNA with length more than 200 nucleotides, which aroused interest of people in recent years. Lots of studies have confirmed that human genome contains many thousands of lncRNAs which exert great influence over some critical regulators of cellular process. With the advent of high-throughput sequencing technologies, a great quantity of sequences is waiting for exploitation. Thus, many programs are developed to distinguish differences between coding and long noncoding transcripts. Different programs are generally designed to be utilised under different circumstances and it is sensible and practical to select an appropriate method according to a certain situation. In this review, several popular methods and their advantages, disadvantages, and application scopes are summarised to assist people in employing a suitable method and obtaining a more reliable result.


September 22, 2019  |  

Emergence, retention and selection: A trilogy of origination for functional de novo proteins from ancestral lncRNAs in primates.

While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.


September 22, 2019  |  

Analyses of alternative polyadenylation: from old school biochemistry to high-throughput technologies.

Alternations in usage of polyadenylation sites during transcription termination yield transcript isoforms from a gene. Recent findings of transcriptome-wide alternative polyadenylation (APA) as a molecular response to changes in biology position APA not only as a molecular event of early transcriptional termination but also as a cellular regulatory step affecting various biological pathways. With the development of high-throughput profiling technologies at a single nucleotide level and their applications targeted to the 3′-end of mRNAs, dynamics in the landscape of mRNA 3′-end is measureable at a global scale. In this review, methods and technologies that have been adopted to study APA events are discussed. In addition, various bioinformatics algorithms for APA isoform analysis using publicly available RNA-seq datasets are introduced. [BMB Reports 2017; 50(4): 201-207].


September 22, 2019  |  

Ensembl 2018

The Ensembl project has been aggregating, processing, integrating and redistributing genomic datasets since the initial releases of the draft human genome, with the aim of accelerating genomics research through rapid open distribution of public data. Large amounts of raw data are thus transformed into knowledge, which is made available via a multitude of channels, in particular our browser (http://www.ensembl.org). Over time, we have expanded in multiple directions. First, our resources describe multiple fields of genomics, in particular gene annotation, comparative genomics, genetics and epigenomics. Second, we cover a growing number of genome assemblies; Ensembl Release 90 contains exactly 100. Third, our databases feed simultaneously into an array of services designed around different use cases, ranging from quick browsing to genome-wide bioinformatic analysis. We present here the latest developments of the Ensembl project, with a focus on managing an increasing number of assemblies, supporting efforts in genome interpretation and improving our browser.


September 22, 2019  |  

The small peptide world in long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are a group of transcripts that are longer than 200 nucleotides (nt) without coding potential. Over the past decade, tens of thousands of novel lncRNAs have been annotated in animal and plant genomes because of advanced high-throughput RNA sequencing technologies and with the aid of coding transcript classifiers. Further, a considerable number of reports have revealed the existence of stable, functional small peptides (also known as micropeptides), translated from lncRNAs. In this review, we discuss the methods of lncRNA classification, the investigations regarding their coding potential and the functional significance of the peptides they encode.


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