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-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.

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.

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.

The generation of direction selectivity in the auditory system.

Both human speech and animal vocal signals contain frequency-modulated (FM) sounds. Although central auditory neurons that selectively respond to the direction of frequency modulation are known, the synaptic mechanisms underlying the generation of direction selectivity (DS) remain elusive. Here we show the emergence of DS neurons in the inferior colliculus by mapping the three major subcortical auditory nuclei. Cell-attached recordings reveal a highly reliable and precise firing of DS neurons to FM sweeps in a preferred direction. By using in vivo whole-cell current-clamp and voltage-clamp recordings, we found that the synaptic inputs to DS neurons are not direction selective, but temporally reversed excitatory and inhibitory synaptic inputs are evoked in response to opposing directions of FM sweeps. The construction of such temporal asymmetry, resulting DS, and its topography can be attributed to the spectral disparity of the excitatory and the inhibitory synaptic tonal receptive fields.