Curation, inference, and assessment of a globally reconstructed gene regulatory network for Streptomyces coelicolor.

Streptomyces coelicolor A3(2) is a model microorganism for the study of Streptomycetes, antibiotic production, and secondary metabolism in general. Even though S. coelicolor has an outstanding variety of regulators among bacteria, little effort to globally study its transcription has been made. We manually curated 29 years of literature and databases to assemble a meta-curated experimentally-validated gene regulatory network (GRN) with 5386 genes and 9707 regulatory interactions (~ 41% of the total expected interactions). This provides the most extensive and up-to-date reconstruction available for the regulatory circuitry of this organism. Only ~ 6% (534/9707) are supported by experiments confirming the binding of the transcription factor to the upstream region of the target gene, the so-called "strong" evidence. While for the remaining interactions there is no confirmation of direct binding. To tackle network incompleteness, we performed network inference using several methods (including two proposed here) for motif identification in DNA sequences and GRN inference from transcriptomics. Further, we contrasted the structural properties and functional architecture of the networks to assess the reliability of the predictions, finding the inference from DNA sequence data to be the most trustworthy approach. Finally, we show two applications of the inferred and the curated networks. The inference allowed us to propose novel transcription factors for the key Streptomyces antibiotic regulatory proteins (SARPs). The curated network allowed us to study the conservation of the system-level components between S. coelicolor and Corynebacterium glutamicum. There we identified the basal machinery as the common signature between the two organisms. The curated networks were deposited in Abasy Atlas ( https://abasy.ccg.unam.mx/ ) while the inferences are available as Supplementary Material.

Human SARS-CoV-2 has evolved to increase U content and reduce genome size.

Infections caused by SARS-CoV-2 have brought great harm to human health. After transmission for over two years, SARS-CoV-2 has diverged greatly and formed dozens of different lineages. Understanding the trend of its genome evolution could help foresee difficulties in controlling transmission of the virus. In this study, we conducted an extensive monthly survey and in-depth analysis on variations of nucleotide, amino acid and codon numbers in 311,260 virus samples collected till January 2022. The results demonstrate that the evolution of SARS-CoV-2 is toward increasing U-content and reducing genome-size. C, G and A to U mutations have all contributed to this U-content increase. Mutations of C, G and A at codon position 1, 2 or 3 have no significant difference in most SARS-CoV-2 lineages. Current viruses are more cryptic and more efficient in replication, and are thus less virulent yet more infectious. Delta and Omicron variants have high mutability over other lineages, bringing new threat to human health. This trend of genome evolution may provide a clue for tracing the origin of SARS-CoV-2, because ancestral viruses should have lower U-content and probably bigger genome-size.

Identification of human gene research articles with wrongly identified nucleotide sequences.

Nucleotide sequence reagents underpin molecular techniques that have been applied across hundreds of thousands of publications. We have previously reported wrongly identified nucleotide sequence reagents in human research publications and described a semi-automated screening tool Seek & Blastn to fact-check their claimed status. We applied Seek & Blastn to screen >11,700 publications across five literature corpora, including all original publications in Gene from 2007 to 2018 and all original open-access publications in Oncology Reports from 2014 to 2018. After manually checking Seek & Blastn outputs for >3,400 human research articles, we identified 712 articles across 78 journals that described at least one wrongly identified nucleotide sequence. Verifying the claimed identities of >13,700 sequences highlighted 1,535 wrongly identified sequences, most of which were claimed targeting reagents for the analysis of 365 human protein-coding genes and 120 non-coding RNAs. The 712 problematic articles have received >17,000 citations, including citations by human clinical trials. Given our estimate that approximately one-quarter of problematic articles may misinform the future development of human therapies, urgent measures are required to address unreliable gene research articles.

Impact of high platelet turnover on the platelet transcriptome: Results from platelet RNA-sequencing in patients with sepsis.

BACKGROUND: Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. METHODS: Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. RESULTS: IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3-8.7] % vs. 3.6 [2.6-4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. CONCLUSIONS: Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.

Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity.

The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. To systematically explore the sequence determinants governing CRISPR/Cas9 specificity, here we describe a dual-target system to measure the relative cleavage rate between off- and on-target sequences (off-on ratios) of 1902 gRNAs on 13,314 synthetic target sequences, and reveal a set of sequence rules involving 2 factors in off-targeting: 1) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context; 2) an "epistasis-like" combinatorial effect of multiple mismatches, which are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. These sequence rules lead to the development of MOFF, a model-based predictor of Cas9-mediated off-target effects. Moreover, the "epistasis-like" combinatorial effect suggests a strategy of allele-specific genome editing using mismatched guides. With the aid of MOFF prediction, this strategy significantly improves the selectivity and expands the application domain of Cas9-based allele-specific editing, as tested in a high-throughput allele-editing screen on 18 cancer hotspot mutations.

Why sequence all eukaryotes?

Life on Earth has evolved from initial simplicity to the astounding complexity we experience today. Bacteria and archaea have largely excelled in metabolic diversification, but eukaryotes additionally display abundant morphological innovation. How have these innovations come about and what constraints are there on the origins of novelty and the continuing maintenance of biodiversity on Earth? The history of life and the code for the working parts of cells and systems are written in the genome. The Earth BioGenome Project has proposed that the genomes of all extant, named eukaryotes-about 2 million species-should be sequenced to high quality to produce a digital library of life on Earth, beginning with strategic phylogenetic, ecological, and high-impact priorities. Here we discuss why we should sequence all eukaryotic species, not just a representative few scattered across the many branches of the tree of life. We suggest that many questions of evolutionary and ecological significance will only be addressable when whole-genome data representing divergences at all of the branchings in the tree of life or all species in natural ecosystems are available. We envisage that a genomic tree of life will foster understanding of the ongoing processes of speciation, adaptation, and organismal dependencies within entire ecosystems. These explorations will resolve long-standing problems in phylogenetics, evolution, ecology, conservation, agriculture, bioindustry, and medicine.

Standards recommendations for the Earth BioGenome Project.

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

Green plant genomes: What we know in an era of rapidly expanding opportunities.

Green plants play a fundamental role in ecosystems, human health, and agriculture. As de novo genomes are being generated for all known eukaryotic species as advocated by the Earth BioGenome Project, increasing genomic information on green land plants is essential. However, setting standards for the generation and storage of the complex set of genomes that characterize the green lineage of life is a major challenge for plant scientists. Such standards will need to accommodate the immense variation in green plant genome size, transposable element content, and structural complexity while enabling research into the molecular and evolutionary processes that have resulted in this enormous genomic variation. Here we provide an overview and assessment of the current state of knowledge of green plant genomes. To date fewer than 300 complete chromosome-scale genome assemblies representing fewer than 900 species have been generated across the estimated 450,000 to 500,000 species in the green plant clade. These genomes range in size from 12 Mb to 27.6 Gb and are biased toward agricultural crops with large branches of the green tree of life untouched by genomic-scale sequencing. Locating suitable tissue samples of most species of plants, especially those taxa from extreme environments, remains one of the biggest hurdles to increasing our genomic inventory. Furthermore, the annotation of plant genomes is at present undergoing intensive improvement. It is our hope that this fresh overview will help in the development of genomic quality standards for a cohesive and meaningful synthesis of green plant genomes as we scale up for the future.

Longitudinal clonal dynamics of HIV-1 latent reservoirs measured by combination quadruplex polymerase chain reaction and sequencing.

HIV-1 infection produces a long-lived reservoir of latently infected CD4+ T cells that represents the major barrier to HIV-1 cure. The reservoir contains both intact and defective proviruses, but only the proviruses that are intact can reinitiate infection upon cessation of antiretroviral therapy (ART). Here we combine four-color quantitative PCR and next-generation sequencing (Q4PCR) to distinguish intact and defective proviruses and measure reservoir content longitudinally in 12 infected individuals. Q4PCR differs from other PCR-based methods in that the amplified proviruses are sequence verified as intact or defective. Samples were collected systematically over the course of up to 10 y beginning shortly after the initiation of ART. The size of the defective reservoir was relatively stable with minimal decay during the 10-y observation period. In contrast, the intact proviral reservoir decayed with an estimated half-life of 4.9 y. Nevertheless, both intact and defective proviral reservoirs are dynamic. As a result, the fraction of intact proviruses found in expanded clones of CD4+ T cells increases over time with a concomitant decrease in overall reservoir complexity. Thus, reservoir decay measurements by Q4PCR are quantitatively similar to viral outgrowth assay (VOA) and intact proviral DNA PCR assay (IPDA) with the addition of sequence information that distinguishes intact and defective proviruses and informs reservoir dynamics. The data are consistent with the notion that intact and defective proviruses are under distinct selective pressure, and that the intact proviral reservoir is progressively enriched in expanded clones of CD4+ T cells resulting in diminishing complexity over time.

Lipid membranes modulate the activity of RNA through sequence-dependent interactions.

RNA is a ubiquitous biomolecule that can serve as both catalyst and information carrier. Understanding how RNA bioactivity is controlled is crucial for elucidating its physiological roles and potential applications in synthetic biology. Here, we show that lipid membranes can act as RNA organization platforms, introducing a mechanism for riboregulation. The activity of R3C ribozyme can be modified by the presence of lipid membranes, with direct RNA-lipid interactions dependent on RNA nucleotide content, base pairing, and length. In particular, the presence of guanine in short RNAs is crucial for RNA-lipid interactions, and G-quadruplex formation further promotes lipid binding. Lastly, by artificially modifying the R3C substrate sequence to enhance membrane binding, we generated a lipid-sensitive ribozyme reaction with riboswitch-like behavior. These findings introduce RNA-lipid interactions as a tool for developing synthetic riboswitches and RNA-based lipid biosensors and bear significant implications for RNA world scenarios for the origin of life.

Range expansion and complete mitochondrial genome of the highfin blenny (Lupinoblennius nicholsi).

BACKGROUND: The highfin blenny, Lupinoblennius nicholsi, is a marine fish species reported in reef and rocky inshore habitats with a disjunct distribution in the southern Gulf of Mexico. Overall, there are very few studies on this species and there is a scarcity of molecular resources for genetic comparisons. We set out to report the first mitochondrial genome for L. nicholsi and report a range expansion for the species. METHODS AND RESULTS: An individual of L. nicholsi was collected from the coast of Dauphin Island, Alabama. The mitochondrial genome was sequenced, assembled, and annotated. The fragment corresponding to cytochrome oxidase I (COI) was used to compare this sample to other cryptobenthic species of the Atlantic. Finding a mature individual in the coast of Alabama implies this species has a continuous distribution throughout the northern Gulf of Mexico. The mitochondrial genome of L. nicholsi is 16,416 bp in length and comprised of 13 protein coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding D-loop. Comparisons using COI support the species is L. nicholsi and separate it from other cryptobenthic fishes found in the area. CONCLUSIONS: This study represents the first mitochondrial genome for this L. nicholsi, serving as a reference for future comparative studies with marine fishes. By reporting the range expansion of this species, this study provides insights on the fish diversity of the Gulf of Mexico.

Molecular and biological characterization of citrus bent leaf viroid from Malaysia.

BACKGROUND: A 328-nucleotide variant of citrus bent leaf viroid (CBLVd) was characterized by citrus varieties in Malaysia. After the first report in Malaysia, the emerging CBLVd was detected in five citrus species, namely Citrofortunella microcarpa, Citrus aurantifolia, Citrus hystrix, Citrus maxima, and Citrus sinensis. METHODS AND RESULTS: CBLVd was detected in 23 out of 133 symptomatic samples through RT-PCR. Sequence analysis of the RT-PCR amplicons from this study showed 99-100% sequence identity to the reference CBLVd Jp isolate and CBLVd isolates reported in Malaysia. Inoculation of sap, obtained from a CBLVd positive sample, into 6-month old healthy C. microcarpa seedlings showed symptoms of slight leaf bending, reduced leaf size of matured leaves, and mild mosaic between 4 to 6 months after inoculation. Moreover, the observed symptoms of chlorosis, midvein necrosis, leaf rolling, and smalling of leaves in calamondin, C. microcarpa (Bunge) Wijnands, were not reported in earlier studies and opened a new avenue for the study of symptomology. The mechanical transmissibility of CBLVd in the inoculated seedlings was reconfirmed by RT-PCR assay and sequencing. CONCLUSIONS: Based on the results, the sequence similarity of CBLVd isolates from different areas of Malaysia showed no significant difference among each other and the reference isolate. The CBLVd is mechanically transmissible and could produce variable symptoms in different hosts.

Comparison between various biosensor methods for human T-lymphotropic virus-1 (HTLV-1) detection.

Due to the drawback of traditional and current diagnostic methods including serological and molecular assays, the development of the rapid and free-PCR techniques can be an alternative technique for the human T-cell lymphotropic virus (HTLV-1) DNA detection sequences. On the other hand, early detection of HTLV-1 prevents two dangerous diseases including Adult T-cell leukemia/lymphoma and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis. The biosensor-based methods are sensitive techniques that can provide new opportunities to detect infectious diseases, particularly in the early stage. This study provides a comparative view among recently designed biosensors for the detection of HTLV-1.

Evolution of the DAN gene family in vertebrates.

The DAN gene family (DAN, Differential screening-selected gene Aberrant in Neuroblastoma) is a group of genes that is expressed during development and plays fundamental roles in limb bud formation and digitation, kidney formation and morphogenesis and left-right axis specification. During adulthood the expression of these genes are associated with diseases, including cancer. Although most of the attention to this group of genes has been dedicated to understanding its role in physiology and development, its evolutionary history remains poorly understood. Thus, the goal of this study is to investigate the evolutionary history of the DAN gene family in vertebrates, with the objective of complementing the already abundant physiological information with an evolutionary context. Our results recovered the monophyly of all DAN gene family members and divide them into five main groups. In addition to the well-known DAN genes, our phylogenetic results revealed the presence of two new DAN gene lineages; one is only retained in cephalochordates, whereas the other one (GREM3) was only identified in cartilaginous fish, holostean fish, and coelacanth. According to the phyletic distribution of the genes, the ancestor of gnathostomes possessed a repertoire of eight DAN genes, and during the radiation of the group GREM1, GREM2, SOST, SOSTDC1, and NBL1 were retained in all major groups, whereas, GREM3, CER1, and DAND5 were differentially lost.

Analysis of opsin gene family of Crimson snapper (Lutjanus erythropterus).

Opsin is a fellow of the G protein-coupled receptors (GPCRs) superfamily. It can be divided into visual and non-visual opsin according to whether it is directly involved in visual imaging. Opsin plays an important role in visual image formation and the regulation of non-image forming functions such as circadian entrainment in the growth, development and evolution of fish. Crimson snapper belongs to Perciforme mainly found in the Indo-West Pacific and the South China Sea. It is one of the most influential economic fishes in the South China Sea. In order to study the existence and expression of opsin gene in Crimson snapper, we sequenced the genome and tissue sample transcriptome of Crimson snapper. In this study, 32 opsin genes were identified from the genome of Crimson snapper. The length of these genes ranged from 1061 bp to 86203 bp and were distributed on 15 different chromosomes. The analysis of opsin gene family of Crimson snapper showed that the sws2 had two extra copies as compared with that of Zebrafish. Domain and motif analysis revealed that all the 32 opsin genes have seven-(pass)-transmembrane domain receptors (7TM receptors) each, and the opsin family contained 10 common motifs. The expression level of opsin gene, confirmed by RT-qPCR, was analyzed by using nine tissues transcriptome databases of Crimson snapper. The results showed that almost all opsin genes were highly expressed in the retina and brain, except opn7a and opn7b which were expressed in intestine and red skin, and almost no expression in other tissues. Our results provide a comprehensive basic knowledge for the opsin gene family of Crimson snapper, which has significance for the study of the function of opsin in Lutjanidaes.

The phylogenic position of aschiphasmatidae in euphasmatodea based on mitochondrial genomic evidence.

The mitochondrial genome (mitogenome) has been regarded as significant source of data to better understand the phylogenetic relationships within the Euphasmatodea, but no mitogenome in Aschiphasmatoidea has been sequenced to date. In this study, two mitogenomes of Orthomeria smaragdinum and Nanhuaphasma hamicercum of Aschiphasmatidae were sequenced and annotated for the first time. The same mitochondrial gene rearrangement structure was present in the two mitogenomes sequenced, showing as the translocation of tRNA-Arg and tRNA-Asn, which conformed to the tandem duplication-random loss and could be used as a possible synapomorphy for Aschiphasmatidae. The phylogenetic results based on the maximum likelihood (ML) and bayesian inference (BI) methods both showed that Aschiphasmatidae and Neophasmatodea in Euphasmatodea are sister taxa. Although the monophyly of Oriophasmata, Occidophasmata, Diapheromeridae, Phasmatidae, Lonchodidae and Bacilloidea has not been solved, the monophyly of Neophasmatodea and Phyllioidea was well supported.

A Conserved Histophilus somni 23S Intervening Sequence Yields Functional, Fragmented 23S rRNA.

Histophilus somni is a Gram-negative bacterial organism that acts as an opportunistic pathogen and is a fastidious member of the Pasteurellaceae family associated with diseases of respiratory, reproductive, cardiac, and other tissues of ruminants. We identified an intervening sequence (IVS) embedded in all five copies of the 23S rRNA gene in the closed genome sequence of the H. somni isolate USDA-ARS-USMARC-63250 that may play an important role in affecting the biology of the organism. Sequencing the RNA from this isolate shows that most of the IVS is cleaved from the transcript, resulting in independent fragments of this structural rRNA that remain functional within the bacterial ribosome. The IVS lies between positions 1170 and 1278 bp of the 3,017-bp gene and exhibits self-complementarity between its 5' and 3' ends that predicts a stem-loop structure interrupting helix-45 in the transcribed 23S rRNA. Excision removes a 94-nucleotide (nt) stem-loop structure that displays an unusual 1-nt 3' end overhang instead of the more typical 2-nt overhang commonly observed at the ends of other excised IVS stem-loops. A comparison with genomes of other H. somni isolates indicates that this IVS is highly conserved, with 31 of 32 complete genomes having similar interruptions of canonical 23S rRNA genes. The potential biological effects of either the released IVS or the fragmentation of the functional 23S rRNA are unknown, but fragmentation may enhance rRNA degradation in ways that contribute to the regulation of gene expression. IMPORTANCE The genome biology underlying H. somni virulence, pathogenicity, environmental adaptability, and broad tissue tropism is understood poorly. We identified a novel H. somni 109-nt IVS stem-loop structure, of which the central portion is excised from the 23S rRNA transcript, resulting in the fragmentation of this rRNA in the H. somni isolate USDA-ARS-USMARC-63250 and the release of a 94-nt structured RNA of unknown function. We determined that this peculiar rRNA biology is widespread among sequenced H. somni isolates, suggesting it has importance to organism biology. The fragmented 23S rRNA molecules remain functional in the ribosome, given that the isolate grows in culture. The structured excised portion of the IVS, presumably due to the action of the endoribonuclease III, has an unusual 1-nt 3' end overhang. This newly discovered H. somni 23S rRNA fragmentation may enhance rRNA degradation providing a previously unrecognized avenue for regulating H. somni biological processes.

VarGenius-HZD Allows Accurate Detection of Rare Homozygous or Hemizygous Deletions in Targeted Sequencing Leveraging Breadth of Coverage.

Homozygous deletions (HDs) may be the cause of rare diseases and cancer, and their discovery in targeted sequencing is a challenging task. Different tools have been developed to disentangle HD discovery but a sensitive caller is still lacking. We present VarGenius-HZD, a sensitive and scalable algorithm that leverages breadth-of-coverage for the detection of rare homozygous and hemizygous single-exon deletions (HDs). To assess its effectiveness, we detected both real and synthetic rare HDs in fifty exomes from the 1000 Genomes Project obtaining higher sensitivity in comparison with state-of-the-art algorithms that each missed at least one event. We then applied our tool on targeted sequencing data from patients with Inherited Retinal Dystrophies and solved five cases that still lacked a genetic diagnosis. We provide VarGenius-HZD either stand-alone or integrated within our recently developed software, enabling the automated selection of samples using the internal database. Hence, it could be extremely useful for both diagnostic and research purposes.

Mitochondrial Genomes of the Genus Claassenia (Plecoptera: Perlidae) and Phylogenetic Assignment to Subfamily Perlinae.

Mitochondrial genomes of three stoneflies, e.g., Claassenia magna Wu, 1948, Claassenia sp. 2 and Claassenia xucheni Chen, 2019 were sequenced in this study with 15,774, 15,777 and 15,746 bp in length, respectively. Each mitogenome contained 37 genes including 22 tRNAs, two ribosomal RNAs, 13 protein-coding genes (PCGs), and a noncoding control region (CR). In general, standard ATN start and TAN termination codons were evident in the PCGs. Although the dihydrouridine arm was absent in trnSer, the remaining 21 tRNAs displayed the characteristic cloverleaf secondary structure. Stem-loop structures were identified in the CRs of all three mitogenomes, but tandem repeats were only apparent in Claassenia xucheni. The mitogenomes of three Claassenia species were analyzed and compared with mitogenomes in 21 other stoneflies from the Perlidae and three Euholognatha species (Rhopalopsole bulbifera, Capnia zijinshana and Amphinemura longispina) as outgroups. Phylogenetic analyses using maximum likelihood and Bayesian inference. Phylogenetic analysis supported that Claassenia was recovered as the sister group of other Perlinae and Claassenia+Perlinae emerged from the paraphyletic Acroneuriinae. The final results supported that Claassenia was classified into subfamily Perlinae and proposed Claassenia represent a transitional group of the subfamilies Acroneuriinae and Perlinae. This study provided new molecular evidence for exploring the debatable taxonomic position of the genus Claassenia in Perlidae.