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Background: Early sequencing and quick analysis of the SARS-CoV-2 genome have contributed to the understanding of the dynamics of COVID-19 epidemics and in designing countermeasures at a global level., Objective: Amplicon-based next-generation sequencing (NGS) methods are widely used to sequence the SARS-CoV-2 genome and to identify novel variants that are emerging in rapid succession as well as harboring multiple deletions and amino acid-changing mutations., Methods: To facilitate the analysis of NGS sequencing data obtained from amplicon-based sequencing methods, here, we propose an easy-to-use SARS-CoV-2 genome assembler: the Easy-to-use SARS-CoV-2 Assembler (ESCA) pipeline., Results: Our results have shown that ESCA could perform high-quality genome assembly from Ion Torrent and Illumina raw data and help the user in easily correct low-coverage regions. Moreover, ESCA includes the possibility of comparing assembled genomes of multisample runs through an easy table format., Conclusions: In conclusion, ESCA automatically furnished a variant table output file, fundamental to rapidly recognizing variants of interest. Our pipeline could be a useful method for obtaining a complete, rapid, and accurate analysis even with minimal knowledge in bioinformatics., Competing Interests: Conflicts of Interest: None declared., (©Martina Rueca, Emanuela Giombini, Francesco Messina, Barbara Bartolini, Antonino Di Caro, Maria Rosaria Capobianchi, Cesare EM Gruber. Originally published in JMIR Bioinformatics and Biotechnology (https://bioinform.jmir.org), 14.03.2022.)

BackgroundCrimean-Congo haemorrhagic fever virus (CCHFV) is considered an emerging infectious disease threat in the European Union. Since 2000, the incidence and geographic range of confirmed CCHF cases have markedly increased, following changes in the distribution of its main vector, Hyalomma ticks.AimsTo review scientific literature and collect experts' opinion to analyse relevant aspects of the laboratory management of human CCHF cases and any exposed contacts, as well as identify areas for advancement of international collaborative preparedness and laboratory response plans.MethodsWe conducted a literature review on CCHF molecular diagnostics through an online search. Further, we obtained expert opinions on the key laboratory aspects of CCHF diagnosis. Consulted experts were members of two European projects, EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level) and EVD-LabNet (Emerging Viral Diseases-Expert Laboratory Network).ResultsConsensus was reached on relevant and controversial aspects of CCHF disease with implications for laboratory management of human CCHF cases, including biosafety, diagnostic algorithm and advice to improve lab capabilities. Knowledge on the diffusion of CCHF can be obtained by promoting syndromic approach to infectious diseases diagnosis and by including CCHFV infection in the diagnostic algorithm of severe fevers of unknown origin.ConclusionNo effective vaccine and/or therapeutics are available at present so outbreak response relies on rapid identification and appropriate infection control measures. Frontline hospitals and reference laboratories have a crucial role in the response to a CCHF outbreak, which should integrate laboratory, clinical and public health responses.

Our objectives for this study were to understand the biological basis of meat tenderness and to provide an overview of the gene expression profiles related to meat quality as a tool for selection. Through deep mRNA sequencing, we analyzed gene expression in muscle tissues of two Italian cattle breeds: Maremmana and Chianina. We uncovered several differentially expressed genes that encode for proteins belonging to a family of tripartite motif proteins, which are involved in growth, cell differentiation and apoptosis, such as TRIM45, or play an essential role in regulating skeletal muscle differentiation and the regeneration of adult skeletal muscle, such as TRIM32. Other differentially expressed genes (SCN2B, SLC9A7 and KCNK3) emphasize the involvement of potassium-sodium pumps in tender meat. By mapping splice junctions in RNA-Seq reads, we found significant differences in gene isoform expression levels. The PRKAG3 gene, which is involved in the regulation of energy metabolism, showed four isoforms that were differentially expressed. This distinct pattern of PRKAG3 gene expression could indicate impaired glycogen storage in skeletal muscle, and consequently, this gene very likely has a role in the tenderization process. Furthermore, with this deep RNA-sequencing, we captured a high number of expressed SNPs, for example, we found 1462 homozygous SNPs showing the alternative allele with a 100% frequency when comparing tender and tough meat. SNPs were then classified into categories by their position and also by their effect on gene coding (174 non-synonymous polymorphisms) based on the available UMD_3.1 annotations., (© 2016 Stichting International Foundation for Animal Genetics.)

Chianina and Maremmana breeds play an important role in the Italian cattle meat market. The Chianina breed is an ancient breed principally raised for draught. Now this breed is the worldwide recognized producer of top quality beef, tasteful and tender, specifically the famous "Florentine steak". The Maremmana characterized by a massive skeletal structure, is a rustic cattle breed selected for adaptability to the marshy land of the Maremma region. We used a high throughput mRNA sequencing to analyze gene expression in muscle tissues of two Italian cattle breeds, Maremmana (MM) and Chianina (CN) with different selection history. We aim to examine the specific genetic contribution of each breed to meat production and quality, comparing the skeletal muscle tissue from Maremmana and Chianina. Most of the differentially expressed genes were grouped in the Glycolysis/Gluconeogenesis pathways. The rate and the extent of post-mortem energy metabolism have a critical effect on the conversion of muscle to meat. Furthermore, we aim at discovering the differences in nucleotide variation between the two breeds which might be attributable to the different history of selection/divergence. In this work we could emphasize the involvement of pathways of post-mortem energy metabolism. Moreover, we detected a collection of coding SNPs which could offer new genomic resources to improve phenotypic selection in livestock breeding program.

We report the complete genome sequence of Ebola virus from a health worker linked to a cluster of cases occurring in the fishing community of Aberdeen, Sierra Leone (February 2015), which were characterized by unusually severe presentation. The sequence, clustering in the SL subclade 3.2.4, harbors mutations potentially relevant for pathogenesis., (Copyright © 2015 Capobianchi et al.)

Here, we report the complete genome sequence of an Ebola virus (EBOV) isolated from a health worker repatriated from Sierra Leone to Italy in November 2014. The sequence, clustering in clade 3 of the Sierra Leone sequences, was analyzed with respect to mutations possibly affecting diagnostic and therapeutic targets as well as virulence., (Copyright © 2015 Castilletti et al.)

Background: Bovine Spastic Paresis (BSP) is a neuromuscular disorder which affects both male and female cattle. BSP is characterized by spastic contraction and overextension of the gastrocnemious muscle of one or both limbs and is associated with a scarce increase in body weight. This disease seems to be caused by an autosomal and recessive gene, with incomplete penetration, although no genes clearly involved with its onset have been so far identified. We employed cDNA microarrays to identify metabolic pathways affected by BSP in Romagnola cattle breed. Investigation of those pathways at the genome level can help to understand this disease., Results: Microarray analysis of control and affected individuals resulted in 268 differentially expressed genes. These genes were subjected to KEGG pathway functional clustering analysis, revealing that they are predominantly involved in Cell Communication, Signalling Molecules and Interaction and Signal Transduction, Diseases and Nervous System classes. Significantly enriched KEGG pathway's classes for the differentially expressed genes were calculated; interestingly, all those significantly under-expressed in the affected samples are included in Neurodegenerative Diseases. To identify genome locations possibly harbouring gene(s) involved in the disease, the chromosome distribution of the differentially expressed genes was also investigated., Conclusions: The cDNA microarray we used in this study contains a brain library and, even if carrying an incomplete transcriptome representation, it has proven to be a valuable tool allowing us to add useful and new information to a poorly studied disease. By using this tool, we examined nearly 15000 transcripts and analysed gene pathways affected by the disease. Particularly, our data suggest also a defective glycinergic synaptic transmission in the development of the disease and an alteration of calcium signalling proteins. We provide data to acquire knowledge of a genetic disease for which literature still presents poor results and that could be further and specifically analysed in the next future. Moreover this study, performed in livestock, may also harbour molecular information useful for understanding human diseases.

Since its start, the Mammalian Gene Collection (MGC) has sought to provide at least one full-protein-coding sequence cDNA clone for every human and mouse gene with a RefSeq transcript, and at least 6200 rat genes. The MGC cloning effort initially relied on random expressed sequence tag screening of cDNA libraries. Here, we summarize our recent progress using directed RT-PCR cloning and DNA synthesis. The MGC now contains clones with the entire protein-coding sequence for 92% of human and 89% of mouse genes with curated RefSeq (NM-accession) transcripts, and for 97% of human and 96% of mouse genes with curated RefSeq transcripts that have one or more PubMed publications, in addition to clones for more than 6300 rat genes. These high-quality MGC clones and their sequences are accessible without restriction to researchers worldwide.

We describe a collection of expressed sequence tags (ESTs) for Saccoglossus kowalevskii, a direct-developing hemichordate valuable for evolutionary comparisons with chordates. The 202,175 ESTs represent 163,633 arrayed clones carrying cDNAs prepared from embryonic libraries, and they assemble into 13,677 continuous sequences (contigs), leaving 10,896 singletons (excluding mitochondrial sequences). Of the contigs, 53% had significant matches when BLAST was used to query the NCBI databases (< or = 10(-10)), as did 51% of the singletons. Contigs most frequently matched sequences from amphioxus (29%), chordates (67%), and deuterostomes (87%). From the clone array, we isolated 400 full-length sequences for transcription factors and signaling proteins of use for evolutionary and developmental studies. The set includes sequences for fox, pax, tbx, hox, and other homeobox-containing factors, and for ligands and receptors of the TGFbeta, Wnt, Hh, Delta/Notch, and RTK pathways. At least 80% of key sequences have been obtained, when judged against gene lists of model organisms. The median length of these cDNAs is 2.3 kb, including 1.05 kb of 3' untranslated region (UTR). Only 30% are entirely matched by single contigs assembled from ESTs. We conclude that an EST collection based on 150,000 clones is a rich source of sequences for molecular developmental work, and that the EST approach is an efficient way to initiate comparative studies of a new organism.

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, we examined the hemichordate Saccoglossus kowalevskii and studied the expression of orthologs of genes that are involved in patterning the chordate central nervous system. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. We propose that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage.

To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of approximately 3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.

We have developed a subtractive hybridization procedure based on the hybridization of a single-stranded phagemid cDNA library (target) to biotinylated RNA (driver). We have applied this method to fibroblast growth factor (FGF) induced-uninduced mouse brain tumor-derived muscle-like cell. BC3Hl cDNA libraries. After hybridization to a C(o)t value of 1000, cDNAs common to the target and driver populations were subtracted up to 231-fold, whereas several highly induced genes were enriched from 2-15-fold. Interestingly, moderately induced genes (e.g., the 12-fold-induced nur/77 gene) were subtracted even at a low C(o)t value of 50. Therefore, at every C(o)t tested, subtractive hybridization tended to equalize the uninduced and moderately induced common sequences within target populations regardless of the abundance of the gene species. These observations suggest that subtractive hybridization should only be used for identifying target genes that are either uniquely expressed or highly induced.

The production of complete cDNA libraries from minimal amounts of starting material (i.e., mRNA) is a major challenge for cDNA cloning technology. This paper reports the cDNA cloning efficiencies of electroporation and in vitro packaging. These two methods produce a tremendous number of recombinant clones (greater than or equal to 1 x 10(8) clones/micrograms cDNA) although electroporation generates more clones at nearly all cDNA concentrations used in a ligation reaction.

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.