Corrigendum: The molecular basis of differential host responses to avian influenza viruses in avian species with differing susceptibility.

[This corrects the article DOI: 10.3389/fcimb.2023.1067993.].

nf-core/isoseq: simple gene and isoform annotation with PacBio Iso-Seq long-read sequencing.

MOTIVATION: Iso-Seq RNA long-read sequencing enables the identification of full-length transcripts and isoforms, removing the need for complex analysis such as transcriptome assembly. However, the raw sequencing data need to be processed in a series of steps before annotation is complete. Here, we present nf-core/isoseq, a pipeline for automatic read processing and genome annotation. Following nf-core guidelines, the pipeline has few dependencies and can be run on any of platforms. AVAILABILITY AND IMPLEMENTATION: The pipeline is freely available online on the nf-core website (https://nf-co.re/isoseq) and on GitHub (https://github.com/nf-core/isoseq) under MIT License (DOI: 10.5281/zenodo.7116979).

The molecular basis of differential host responses to avian influenza viruses in avian species with differing susceptibility.

Introduction: Highly pathogenic avian influenza (HPAI) viruses, such as H5N1, continue to pose a serious threat to animal agriculture, wildlife and to public health. Controlling and mitigating this disease in domestic birds requires a better understanding of what makes some species highly susceptible (such as turkey and chicken) while others are highly resistant (such as pigeon and goose). Susceptibility to H5N1 varies both with species and strain; for example, species that are tolerant of most H5N1 strains, such as crows and ducks, have shown high mortality to emerging strains in recent years. Therefore, in this study we aimed to examine and compare the response of these six species, to low pathogenic avian influenza (H9N2) and two strains of H5N1 with differing virulence (clade 2.2 and clade 2.3.2.1) to determine how susceptible and tolerant species respond to HPAI challenge. Methods: Birds were challenged in infection trials and samples (brain, ileum and lung) were collected at three time points post infection. The transcriptomic response of birds was examined using a comparative approach, revealing several important discoveries. Results: We found that susceptible birds had high viral loads and strong neuro-inflammatory response in the brain, which may explain the neurological symptoms and high mortality rates exhibited following H5N1 infection. We discovered differential regulation of genes associated with nerve function in the lung and ileum, with stronger differential regulation in resistant species. This has intriguing implications for the transmission of the virus to the central nervous system (CNS) and may also indicate neuro-immune involvement at the mucosal surfaces. Additionally, we identified delayed timing of the immune response in ducks and crows following infection with the more deadly H5N1 strain, which may account for the higher mortality in these species caused by this strain. Lastly, we identified candidate genes with potential roles in susceptibility/resistance which provide excellent targets for future research. Discussion: This study has helped elucidate the responses underlying susceptibility to H5N1 influenza in avian species, which will be critical in developing sustainable strategies for future control of HPAI in domestic poultry.

The swan genome and transcriptome, it is not all black and white.

BACKGROUND: The Australian black swan (Cygnus atratus) is an iconic species with contrasting plumage to that of the closely related northern hemisphere white swans. The relative geographic isolation of the black swan may have resulted in a limited immune repertoire and increased susceptibility to infectious diseases, notably infectious diseases from which Australia has been largely shielded. Unlike mallard ducks and the mute swan (Cygnus olor), the black swan is extremely sensitive to highly pathogenic avian influenza. Understanding this susceptibility has been impaired by the absence of any available swan genome and transcriptome information. RESULTS: Here, we generate the first chromosome-length black and mute swan genomes annotated with transcriptome data, all using long-read based pipelines generated for vertebrate species. We use these genomes and transcriptomes to show that unlike other wild waterfowl, black swans lack an expanded immune gene repertoire, lack a key viral pattern-recognition receptor in endothelial cells and mount a poorly controlled inflammatory response to highly pathogenic avian influenza. We also implicate genetic differences in SLC45A2 gene in the iconic plumage of the black swan. CONCLUSION: Together, these data suggest that the immune system of the black swan is such that should any avian viral infection become established in its native habitat, the black swan would be in a significant peril.

The Role of Dendritic Cells in the Host Response to Marek's Disease Virus (MDV) as Shown by Transcriptomic Analysis of Susceptible and Resistant Birds.

Despite the successful control of highly contagious tumorigenic Marek's disease (MD) by vaccination, a continuous increase in MD virus (MDV) virulence over recent decades has put emphasis on the development of more MD-resistant chickens. The cell types and genes involved in resistance therefore need to be recognized. The virus is primarily lymphotropic, but research should also focus on innate immunity, as innate immune cells are among the first to encounter MDV. Our previous study on MDV-macrophage interaction revealed significant differences between MHC-congenic lines 61 (MD-resistant) and 72 (MD-susceptible). To investigate the role of dendritic cells (DCs) in MD resistance, bone-marrow-derived DCs from these lines were infected with MDV in vitro. They were then characterized by cell sorting, and the respective transcriptomes analysed by RNA-seq. The differential expression (DE) of genes revealed a strong immune activation in DCs of the susceptible line, although an inherent immune supremacy was shown by the resistant line, including a significant expression of tumour-suppressor miRNA, gga-mir-124a, in line 61 control birds. Enrichment analysis of DE genes revealed high expression of an oncogenic transcription factor, AP-1, in the susceptible line following MDV challenge. This research highlights genes and pathways that may play a role in DCs in determining resistance or susceptibility to MDV infection.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

A high-quality genome and comparison of short- versus long-read transcriptome of the palaearctic duck Aythya fuligula (tufted duck).

BACKGROUND: The tufted duck is a non-model organism that experiences high mortality in highly pathogenic avian influenza outbreaks. It belongs to the same bird family (Anatidae) as the mallard, one of the best-studied natural hosts of low-pathogenic avian influenza viruses. Studies in non-model bird species are crucial to disentangle the role of the host response in avian influenza virus infection in the natural reservoir. Such endeavour requires a high-quality genome assembly and transcriptome. FINDINGS: This study presents the first high-quality, chromosome-level reference genome assembly of the tufted duck using the Vertebrate Genomes Project pipeline. We sequenced RNA (complementary DNA) from brain, ileum, lung, ovary, spleen, and testis using Illumina short-read and Pacific Biosciences long-read sequencing platforms, which were used for annotation. We found 34 autosomes plus Z and W sex chromosomes in the curated genome assembly, with 99.6% of the sequence assigned to chromosomes. Functional annotation revealed 14,099 protein-coding genes that generate 111,934 transcripts, which implies a mean of 7.9 isoforms per gene. We also identified 246 small RNA families. CONCLUSIONS: This annotated genome contributes to continuing research into the host response in avian influenza virus infections in a natural reservoir. Our findings from a comparison between short-read and long-read reference transcriptomics contribute to a deeper understanding of these competing options. In this study, both technologies complemented each other. We expect this annotation to be a foundation for further comparative and evolutionary genomic studies, including many waterfowl relatives with differing susceptibilities to avian influenza viruses.

Clergy as Suicide Prevention Gatekeepers.

801 U.S. Catholic, Jewish and Protestant clergy reported on their suicide gatekeeping activities. Using vignettes, they identified suicide risk and selected interventions for three risk levels. Two-thirds of the sample who provide counseling reported at least one contact from a suicidal person per year. Clergy were significantly more concurrent with experts in identifying risk and selecting interventions with high risk but deviated more from the experts with low and medium risk. Most reported needing more training.

Avian Immunome DB: an example of a user-friendly interface for extracting genetic information.

BACKGROUND: Genomic and genetic studies often require a target list of genes before conducting any hypothesis testing or experimental verification. With the ever-growing number of sequenced genomes and a variety of different annotation strategies, comes the potential for ambiguous gene symbols, making it cumbersome to capture the "correct" set of genes. In this article, we present and describe the Avian Immunome DB (AVIMM) for easy gene property extraction as exemplified by avian immune genes. The avian immune system is characterised by a cascade of complex biological processes underlaid by more than 1000 different genes. It is a vital trait to study particularly in birds considering that they are a significant driver in spreading zoonotic diseases. With the completion of phase II of the B10K ("Bird 10,000 Genomes") consortium's whole-genome sequencing effort, we have included 363 annotated bird genomes in addition to other publicly available bird genome data which serve as a valuable foundation for AVIMM. CONSTRUCTION AND CONTENT: A relational database with avian immune gene evidence from Gene Ontology, Ensembl, UniProt and the B10K consortium has been designed and set up. The foundation stone or the "seed" for the initial set of avian immune genes is based on the well-studied model organism chicken (Gallus gallus). Gene annotations, different transcript isoforms, nucleotide sequences and protein information, including amino acid sequences, are included. Ambiguous gene names (symbols) are resolved within the database and linked to their canonical gene symbol. AVIMM is supplemented by a command-line interface and a web front-end to query the database. UTILITY AND DISCUSSION: The internal mapping of unique gene symbol identifiers to canonical gene symbols allows for an ambiguous gene property search. The database is organised within core and feature tables, which makes it straightforward to extend for future purposes. The database design is ready to be applied to other taxa or biological processes. Currently, the database contains 1170 distinct avian immune genes with canonical gene symbols and 612 synonyms across 363 bird species. While the command-line interface readily integrates into bioinformatics pipelines, the intuitive web front-end with download functionality offers sophisticated search functionalities and tracks the origin for each record. AVIMM is publicly accessible at https://avimm.ab.mpg.de .

Illuminating the dark side of the human transcriptome with long read transcript sequencing.

BACKGROUND: The human transcriptome annotation is regarded as one of the most complete of any eukaryotic species. However, limitations in sequencing technologies have biased the annotation toward multi-exonic protein coding genes. Accurate high-throughput long read transcript sequencing can now provide additional evidence for rare transcripts and genes such as mono-exonic and non-coding genes that were previously either undetectable or impossible to differentiate from sequencing noise. RESULTS: We developed the Transcriptome Annotation by Modular Algorithms (TAMA) software to leverage the power of long read transcript sequencing and address the issues with current data processing pipelines. TAMA achieved high sensitivity and precision for gene and transcript model predictions in both reference guided and unguided approaches in our benchmark tests using simulated Pacific Biosciences (PacBio) and Nanopore sequencing data and real PacBio datasets. By analyzing PacBio Sequel II Iso-Seq sequencing data of the Universal Human Reference RNA (UHRR) using TAMA and other commonly used tools, we found that the convention of using alignment identity to measure error correction performance does not reflect actual gain in accuracy of predicted transcript models. In addition, inter-read error correction can cause major changes to read mapping, resulting in potentially over 6 K erroneous gene model predictions in the Iso-Seq based human genome annotation. Using TAMA's genome assembly based error correction and gene feature evidence, we predicted 2566 putative novel non-coding genes and 1557 putative novel protein coding gene models. CONCLUSIONS: Long read transcript sequencing data has the power to identify novel genes within the highly annotated human genome. The use of parameter tuning and extensive output information of the TAMA software package allows for in depth exploration of eukaryotic transcriptomes. We have found long read data based evidence for thousands of unannotated genes within the human genome. More development in sequencing library preparation and data processing are required for differentiating sequencing noise from real genes in long read RNA sequencing data.

Erratum: Chakraborty, P. et al. Macrophages from Susceptible and Resistant Chicken Lines have Different Transcriptomes following Marek's Disease Virus Infection. Genes 2019, 10, 74.

The authors wish to make the following correction to their paper published in Genes [...].

Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses.

To address the need for sensitive high-throughput assays to analyse avian innate and adaptive immune responses, we developed and validated a highly multiplexed qPCR 96.96 Fluidigm Dynamic Array to analyse the transcription of chicken immune-related genes. This microfluidic system permits the simultaneous analysis of expression of 96 transcripts in 96 samples in 6 nanolitre reactions and the 9,216 reactions are ready for interpretation immediately. A panel of 89 genes was selected from an RNA-seq analysis of the transcriptional response of chicken macrophages, dendritic cells and heterophils to agonists of innate immunity and from published transcriptome data. Assays were confirmed to be highly specific by amplicon sequencing and melting curve analysis and the reverse transcription and preamplification steps were optimised. The array was applied to RNA of various tissues from a commercial line of broiler chickens housed at two different levels of biosecurity. Gut-associated lymphoid tissues, bursa, spleen and peripheral blood leukocytes were isolated and transcript levels for immune-related genes were defined. The results identified blood cells as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially transcribed between birds housed under varying biosecurity levels. Conventional qPCR analysis of three differentially transcribed genes confirmed the results from the multiplex qPCR array. A highly multiplexed qPCR-based platform for evaluation of chicken immune responses has been optimised and validated using samples from commercial chickens. Apart from applications in selective breeding programmes, the array could be used to analyse the complex interplay between the avian immune system and pathogens by including pathogen-specific probes, to screen vaccine responses, and as a predictive tool for immune robustness.

Full-length transcriptome sequencing from multiple tissues of duck, Anas platyrhynchos.

Duck (Anas platyrhynchos), one of the most economically important waterfowl, is an ideal model for studying the immune protection mechanism of birds. An incomplete duck reference genome and very limited availability of full-length cDNAs has hindered the identification of alternatively spliced transcripts and slowed down many basic studies in ducks. We applied PacBio Iso-Seq technologies to multiple tissues from duck for use in transcriptome sequencing. We obtained 199,993 full-length transcripts and comprehensively annotated these transcripts. 23,755 lncRNAs were predicted from all identified transcripts and 35,031 alternative splicing events, which divided into 5 models, were accurately predicted from 3,346 genes. Our data constitute a large increase in the known number of both lncRNA, and alternatively spliced transcripts of duck and plays an important role in improving current genome annotation. In addition, the data will be extremely useful for functional studies in other birds.

Correction: Thoracic aortic calcification across the clinical dysglycemic continuum in a large Asian population free of cardiovascular symptoms.

[This corrects the article DOI: 10.1371/journal.pone.0207089.].

Macrophages from Susceptible and Resistant Chicken Lines have Different Transcriptomes following Marek's Disease Virus Infection.

Despite successful control by vaccination, Marek's disease (MD) has continued evolving to greater virulence over recent years. To control MD, selection and breeding of MD-resistant chickens might be a suitable option. MHC-congenic inbred chicken lines, 61 and 72, are highly resistant and susceptible to MD, respectively, but the cellular and genetic basis for these phenotypes is unknown. Marek's disease virus (MDV) infects macrophages, B-cells, and activated T-cells in vivo. This study investigates the cellular basis of resistance to MD in vitro with the hypothesis that resistance is determined by cells active during the innate immune response. Chicken bone marrow-derived macrophages from lines 61 and 72 were infected with MDV in vitro. Flow cytometry showed that a higher percentage of macrophages were infected in line 72 than in line 61. A transcriptomic study followed by in silico functional analysis of differentially expressed genes was then carried out between the two lines pre- and post-infection. Analysis supports the hypothesis that macrophages from susceptible and resistant chicken lines display a marked difference in their transcriptome following MDV infection. Resistance to infection, differential activation of biological pathways, and suppression of oncogenic potential are among host defense strategies identified in macrophages from resistant chickens.

Thoracic aortic calcification across the clinical dysglycemic continuum in a large Asian population free of cardiovascular symptoms.

Thoracic aortic calcification (TAC) is tightly linked to pathological atherosclerosis and associated with certain cardiovascular diseases. While diabetes mellitus (DM) is known as a coronary heart disease equivalent, we examined the presence of TAC across the dysglycemic spectrum of diabetes mellitus (DM). We consecutively studied 3003 asymptomatic ethnic Asians underwent annual cardiovacular health survey, and further categorized them into: 1) 1760 normo-glycemic, 2) 968 pre-diabetic, and 3) 274 overt DM based on dysglycemic indices and medical histories. Several TAC parameters were assessed using non-contrast multi-detector computed tomography (MDCT), and related to dysglycemic indices or diabetes mellitus status. A remarkably graded increases of adjusted total TAC calcium burden, volume and density were seen across Non-diabetes, Pre-diabetes, and diabetes mellitus categories and positively correlated with all dysglycemic profiles (all p<0.001). Multi-variate logistic and linear regression models demonstrated independent associations between greater TAC density and all dysglycemic indices (Coef: 2.5, 1.4, 6.8 for fasting, postprandial sugar and HbA1c) and diabetes mellitus status (all p<0.05). Furthermore, Receiver-operating characteristic curves (ROC) showed fasting sugar and postprandial sugar set at 103mg/dL and 111mg/dL, separately, with HbA1c set at 5.8% all predict the presence of aortic calcification. Dysglycemic status, even without overt diabetes mellitus, were tighly linked to subclinical, pathological thoracic aortic calcification.

Associations between CT-determined visceral fat burden, hepatic steatosis, circulating white blood cell counts and neutrophil-to-lymphocyte ratio.

Visceral adiposity is associated with cardiovascular disease, an association that may be mediated in part by inflammation. We hypothesized that regional measures of visceral adiposity would associate with commonly obtained clinical measures of immune status. We consecutively studied 3,291 subjects (mean age, 49.8±9.8 years) who underwent an annual cardiovascular risk survey. Peri-cardial (PCF) and thoracic peri-aortic adipose tissue (TAT) volumes were determined by dedicated computed tomography (CT) software (Aquarius 3D Workstation, TeraRecon, San Mateo, CA, USA). Hepatic steatosis was assessed by abdominal ultrasonography. We explored cross-sectional associations between visceral fat measures and high-sensitivity C-reactive protein (hs-CRP), leukocyte counts, and the neutrophil-to-lymphocyte ration (NLR). Among 3,291 study participants, we observed positive linear associations between PCF and TAT, higher degree of hepatic steatosis and hs-CRP, various leukocyte counts, either total and its differential counts, and NLR (all trend p<0.001). Multi-variate linear and logistic regression models showed independent associations between PCF/TAT (ß-Coef: 0.14/0.16, both p<0.05) and total WBC counts, with only TAT further demonstrated significant relations with neutrophil counts and NLR (both p<0.05) and independently identified abnormally high WBC and NLR (Odds ratio: 1.18 & 1.21, both p<0.05). C-statistics showed significant incremental model prediction for abnormally high WBC and NLR (both ΔAUROC<0.05) when TAT was superimposed on traditional cardiovascular risks and biochemical information. Greater visceral adiposity burden and hepatic steatosis may be associated with higher circulating leukocyte counts and markers for atherosclerosis, with more pronounced influences for peri-aortic adiposity. Our data suggested the differential biological impacts for region-specific visceral adiposity.

Lipoprotein lipase reaches the capillary lumen in chickens despite an apparent absence of GPIHBP1.

In mammals, GPIHBP1 is absolutely essential for transporting lipoprotein lipase (LPL) to the lumen of capillaries, where it hydrolyzes the triglycerides in triglyceride-rich lipoproteins. In all lower vertebrate species (e.g., birds, amphibians, reptiles, fish), a gene for LPL can be found easily, but a gene for GPIHBP1 has never been found. The obvious question is whether the LPL in lower vertebrates is able to reach the capillary lumen. Using purified antibodies against chicken LPL, we showed that LPL is present on capillary endothelial cells of chicken heart and adipose tissue, colocalizing with von Willebrand factor. When the antibodies against chicken LPL were injected intravenously into chickens, they bound to LPL on the luminal surface of capillaries in heart and adipose tissue. LPL was released rapidly from chicken hearts with an infusion of heparin, consistent with LPL being located inside blood vessels. Remarkably, chicken LPL bound in a specific fashion to mammalian GPIHBP1. However, we could not identify a gene for GPIHBP1 in the chicken genome, nor could we identify a transcript for GPIHBP1 in a large chicken RNA-seq data set. We conclude that LPL reaches the capillary lumen in chickens - as it does in mammals - despite an apparent absence of GPIHBP1.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.