StartLink and StartLink+: Prediction of Gene Starts in Prokaryotic Genomes

State-of-the-art algorithms of ab initio gene prediction for prokaryotic genomes were shown to be sufficiently accurate. A pair of algorithms would agree on predictions of gene 3′ends. Nonetheless, predictions of gene starts would not match for 15–25% of genes in a genome. This discrepancy is a serious issue that is difficult to be resolved due to the absence of sufficiently large sets of genes with experimentally verified starts. We have introduced StartLink that infers gene starts from conservation patterns revealed by multiple alignments of homologous nucleotide sequences. We also have introduced StartLink+ combining both ab initio and alignment-based methods. The ability of StartLink to predict the start of a given gene is restricted by the availability of homologs in a database. We observed that StartLink made predictions for 85% of genes per genome on average. The StartLink+ accuracy was shown to be 98–99% on the sets of genes with experimentally verified starts. In comparison with database annotations, we observed that the annotated gene starts deviated from the StartLink+ predictions for ∼5% of genes in AT-rich genomes and for 10–15% of genes in GC-rich genomes on average. The use of StartLink+ has a potential to significantly improve gene start annotation in genomic databases.

Women, Administered Insecurity, and Multiple Alignments to Assuage Gender Inequalities in the Brazilian Zika Epidemic

This article deals with the Brazilian Zika epidemic that started in 2015 and became an important event for understanding Gender and Power in health treatment contexts. It discusses a combination of reinforcement of gender normed care and therapeutic itineraries overburdening mothers and the construction of political awareness and practice associated with demands for state services. Working with a concept of administered insecurity elaborated to understand people displaced during the implementation of planned government development policies, it argues that the planned nature of health systems, despite their explicit emphasis on the objective of treating the health of the population, also generate administered insecurity as planned administrators of scarcity. It uses data from a 4-year research project entitled “Doing Ethnography on Care” in Recife, Brazil, to show the multiplicity of contexts that are brought out through the practice of mothers in providing care for their Zika-syndrome stricken children, and how they reflect varying power relations that constantly re-dimension maternity along lines of gender relations in different institutional domains of treatment and research. The severe gender inequality in caring for infants was partially assuaged by multiple alignments made by the mothers and infants promoting dialogue and practice in varied contexts, including interaction with the research team. Family and Community Relations, Mediation, Favors, Accusations, Judicialization, Collective Action, formation of Associations, and learning to produce videos are seen as constructors of complex meaning and practice of mothering in a way in which gender goes beyond the limits of an overburdening practice of care. Gender provides a significant difference for mothers whose knowledge and familiarity of domains of health and health-related provision of services and knowledge, forged in their search for treatment for their child, create and legitimate spaces for the exercise of informed citizenship and a sharp awareness and resistance to practices by the state and other agents who administer insecurity. The final remarks synthesize some of the more important reconfigurations of gender relations and power in the context of the Zika epidemic and alerts to the challenge of the transitory nature of an epidemic and the gradual, and not so gradual, exhaustion of institutional interest for the dilemma of the mothers and also shows the role of anthropological research in promoting gender equality in epidemic contexts.

Molecular and Morphological Data Confirmed First Record of Abbreviata kazakhstanica Markov and Paraskiv, 1956 (Spirurida: Physalopteridea) in Iran

Background: The genus Abbreviata (Spirurida: Physalopteridea) currently contains 47 species. Physalopteridae nematodes infect a large number of vertebrates, including mammals, birds, reptiles and amphibians. The current study is a report of the first morphological and molecular identification of A. kazakhstanica (Spirurida: Physalopteridea) in Pseudopus apodus in Iran. Methods: Eleven road-killed P. apodus, were collected from, Iran during 2016-2018. The nematodes were isolated from stomach. After morphological study, the genomic DNA of the parasites was extracted using CTAB method. The DNA was used for PCR amplification of cytochrome c oxidase subunit I (cox1). The PCR products were sequenced, the sequence data were analyzed and multiple alignments were conducted using the Clustal Omega. Results: After detailed microscopic examination, the A. kazakhstanica was identified. The cox1 sequences confirmed the species of helminth. The new sequences of A. kazakhstanica were submitted to GenBank under the accession number MK578751-2. Conclusion: Regarding the limited data on parasitological status of Iranian reptiles, more specific and comprehensive investigations are needed to identify the parasitic fauna.

S-D614G Mutation Reveals the Euro-America and East-Asia Origin SARS-CoV-2 Virus Spread in Indonesia

COVID-19 is a pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The first case was found in the city of Wuhan, Hubei province, China. The first case in Indonesia was reported in March 2020 and currently there are 0.5 million cases with a death rate of 3.1%. This rapid increase in cases is thought to due to presence of the mutant strain S-D614G, which causes a faster rate of infection and spread. The purpose of this study was to determine the presence of S-D614G mutations in Indonesian samples in order to find the origin of COVID-19 which was spread in Indonesia based on the Spike gene sequences and the RdRp genes from 25 countries, and one control sequence China/Wuhan-Hu-1 obtained from the NCBI and GISAID databases. Mutation analysis was carried out through multiple alignments using BioEdit software. Phylogenetic tree reconstruction using MEGA6 software with the Neighbor Joining method. This study found mutation of S-D614G in one Indonesian sample, namely the Indonesian/SBY9 sample along with 23 samples from Europe, America, and Africa. The phylogenetic tree reconstruction confirmed these findings; the mutated samples were closely related to samples from these continents, while the non-mutated Indonesian samples were closely related to sample from East Asia. These findings indicate that the origin of the SARS-CoV-2 virus in Indonesia possibly came from the East Asia cluster and the European-American cluster.

Comparative genomics and characterization of SARS-CoV-2 P.1 (Gamma) Variant of Concern (VOC) from Amazonas, Brazil

Background: P.1 lineage (Gamma) was first described in the State of Amazonas, northern Brazil, in the end of 2020, and has emerged as a very important variant of concern (VOC) of SARS-CoV-2 worldwide. P.1 has been linked to increased infectivity, higher mortality and immune evasion, leading to reinfections and potentially reduced efficacy of vaccines and neutralizing antibodies. Methods: The samples of 276 patients from the State of Amazonas were sent to a central referral laboratory for sequencing by gold standard techniques, through Illumina MiSeq platform. Both global and regional phylogenetic analyses of the successfully sequenced genomes were conducted through maximum likelihood method. Multiple alignments were obtained including previously obtained unique human SARS-CoV-2 sequences. The evolutionary histories of spike and non-structural proteins from ORF1a of northern genomes were described and their molecular evolution was analyzed for detection of positive (FUBAR, FEL, and MEME) and negative (FEL and SLAC) selective pressures. To further evaluate the possible pathways of evolution leading to the emergence of P.1, we performed specific analysis for copy-choice recombination events. A global phylogenomic analysis with subsampled P.1 and B.1.1.28 genomes was applied to evaluate the relationship among samples. Results: Forty-four samples from the State of Amazonas were successfully sequenced and confirmed as P.1 (Gamma) lineage. In addition to previously described P.1 characteristic mutations, we find evidence of continuous diversification of SARS-CoV-2, as rare and previously unseen P.1 mutations were detected in spike and non-structural protein from ORF1a. No evidence of recombination was found. Several sites were demonstrated to be under positive and negative selection, with various mutations identified mostly in P.1 lineage. According to the Pango assignment, phylogenomic analyses indicate all samples as belonging to the P.1 lineage. Conclusion: P.1 has shown continuous evolution after its emergence. The lack of clear evidence for recombination and the positive selection demonstrated for several sites suggest that this lineage emergence resulted mainly from strong evolutionary forces and progressive accumulation of a favorable signature set of mutations.

Molecular Characterization of the First Alternavirus Identified in Fusarium oxysporum

A novel mycovirus named Fusarium oxysporum alternavirus 1(FoAV1) was identified as infecting Fusarium oxysporum strain BH19, which was isolated from a fusarium wilt diseased stem of Lilium brownii. The genome of FoAV1 contains four double-stranded RNA (dsRNA) segments (dsRNA1, dsRNA 2, dsRNA 3 and dsRNA 4, with lengths of 3.3, 2.6, 2.3 and 1.8 kbp, respectively). Additionally, dsRNA1 encodes RNA-dependent RNA polymerase (RdRp), and dsRNA2- dsRNA3- and dsRNA4-encoded hypothetical proteins (ORF2, ORF3 and ORF4), respectively. A homology BLAST search, along with multiple alignments based on RdRp, ORF2 and ORF3 sequences, identified FoAV1 as a novel member of the proposed family “Alternaviridae”. Evolutionary relation analyses indicated that FoAV1 may be related to alternaviruses, thus dividing the family “Alternaviridae” members into four clades. In addition, we determined that dsRNA4 was dispensable for replication and may be a satellite-like RNA of FoAV1—and could perhaps play a role in the evolution of alternaviruses. Our results provided evidence for potential genera establishment within the proposed family “Alternaviridae”. Additionally, FoAV1 exhibited biological control of Fusarium wilt. Our results also laid the foundations for the further study of mycoviruses within the family “Alternaviridae”, and provide a potential agent for the biocontrol of diseases caused by F. oxysporum.

Comparative Genomics of Copia and Gypsy Retroelements in Three Banana Genomes: A, B, and S Genomes

In plants, the proportion of transposable elements (TEs) is generally dominated by long terminal repeat (LTR) retroelements. Therefore, it significantly impacts on genome expansion and genetic and phenotypic variation, namely Copia and Gypsy. Despite such contribution, TEs characterisation in an important crop such as banana [Musa balbisiana (B genome), Musa acuminata (A genome), and Musa schizocarpa (S genome)] remains poorly understood. This study aimed to compare B, A, and S genomes based on repetitive element proportions and copy numbers and determine the evolutionary relationship of LTR using phylogenetic analysis of the reverse transcriptase (RT) domain. Genome assemblies were acquired from the Banana Genome Hub (banana-genome-hub.southgreen.fr). Repetitive elements were masked by RepeatMasker 4.0.9 before Perl parsing. Phylograms were constructed according to domain analysis using DANTE (Domain-based ANnotation of Transposable Elements), alignments were made using MAFFT 7 (multiple alignments using fast Fourier transform), and trees were inferred using FastTree 2. The trees were inspected using SeaView 4 and visualised with FigTree 1.4.4. We reported that B, A, and S genomes are composed of repetitive elements with 19.38%, 20.78%, and 25.96%, respectively. The elements were identified with dominant proportions in the genome are LTR, in which Copia is more abundant than Gypsy. Based on RT phylogenetic analysis, LTR elements are clustered into 13 ancient lineages in which Sire (Copia) and Reina (Gypsy) are shown to be the most abundant LTR lineages in bananas.

T1TAdb: the database of Type I Toxin-Antitoxin systems

Type I toxin-antitoxin (T1TA) systems constitute a large class of genetic modules with antisense RNA (asRNA)-mediated regulation of gene expression. They are widespread in bacteria and consist of an mRNA coding for a toxic protein and a noncoding asRNA that acts as an antitoxin preventing the synthesis of the toxin by directly basepairing to its cognate mRNA. The co- and post-transcriptional regulation of T1TA systems is intimately linked to RNA sequence and structure, therefore it is essential to have an accurate annotation of the mRNA and asRNA molecules to understand this regulation. However, most T1TA systems have been identified by means of bioinformatic analyses solely based on the toxin protein sequences, and there is no central repository of information on their specific RNA features. Here we present the first database dedicated to type I TA systems, named T1TAdb. It is an open-access web database (https://d-lab.arna.cnrs.fr/t1tadb) with a collection of ~1,900 loci in ~500 bacterial strains in which a toxin-coding sequence has been previously identified. RNA molecules were annotated with a bioinformatic procedure based on key determinants of the mRNA structure and the genetic organization of the T1TA loci. Besides RNA and protein secondary structure predictions, T1TAdb also identifies promoter, ribosome-binding, and mRNA-asRNA interaction sites. It also includes tools for comparative analysis, such as sequence similarity search and computation of structural multiple alignments, which are annotated with covariation information. To our knowledge, T1TAdb represents the largest collection of features, sequences, and structural annotations on this class of genetic modules.

Evolutionary dynamics of piRNA clusters in Drosophila

Small RNAs produced from transposable element (TE) rich sections of the genome, termed piRNA clusters, are a crucial component in the genomic defense against selfish DNA. In animals it is thought the invasion of a TE is stopped when a copy of the TE inserts into a piRNA cluster, triggering the production of cognate small RNAs that silence the TE. Despite this importance for TE control, little is known about the evolutionary dynamics of piRNA clusters, mostly because these repeat rich regions are difficult to assemble and compare. Here we establish a framework for studying the evolution of piRNA clusters quantitatively. Previously introduced quality metrics and a newly developed software for multiple alignments of repeat annotations (Manna) allow us to estimate the level of polymorphism segregating in piRNA clusters and the divergence among homologous piRNA clusters. By studying 20 conserved piRNA clusters in multiple assemblies of four Drosophila species we show that piRNA clusters are evolving rapidly. While 70-80% of the clusters are conserved within species, the clusters share almost no similarity between species as closely related as D. melanogaster and D. simulans. Furthermore, abundant insertions and deletions are segregating within the Drosophila species. We show that the evolution of clusters is mainly driven by large insertions of recently active TEs, and smaller deletions mostly in older TEs. The effect of these forces is so rapid that homologous clusters often do not contain insertions from the same TE families.x

A multiple genome alignment workflow shows the impact of repeat masking and parameter tuning on alignment of functional regions in plants

Alignments of multiple genomes are a cornerstone of comparative genomics, but generating these alignments remains technically challenging and often impractical. We developed the msa_pipeline workflow (https://bitbucket.org/bucklerlab/msa_pipeline) based on the LAST aligner to allow practical and sensitive multiple alignment of diverged plant genomes with minimal user inputs. Our workflow only requires a set of genomes in FASTA format as input. The workflow outputs multiple alignments in MAF format, and includes utilities to help calculate genome-wide conservation scores. As high repeat content and genomic divergence are substantial challenges in plant genome alignment, we also explored the impact of different masking approaches and alignment parameters using genome assemblies of 33 grass species. Compared to conventional masking with RepeatMasker, a k-mer masking approach increased the alignment rate of CDS and non-coding functional regions by 25% and 14% respectively. We further found that default alignment parameters generally perform well, but parameter tuning can increase the alignment rate for non-coding functional regions by over 52% compared to default LAST settings. Finally, by increasing alignment sensitivity from the default baseline, parameter tuning can increase the number of non-coding sites that can be scored for conservation by over 76%.