Circular Sequence Research Articles

Complete genome sequence of the saprophytic actinomycete Prescottella soli DSD51WT, closely related to the multi-host pathogen Prescottella (Rhodococcus) equi.

Prescottella soli strain DSD51WT is an aerobic, non-spore-forming, non-motile actinomycete isolated previously from soil collected from Kyoto Park, Japan, using a resuscitative technique. Here, we report the complete, circular genome sequence of P. soli DSD51WT. We employed a hybrid approach using Illumina and Oxford Nanopore platforms.

Complete genome sequence of polylactic acid degrading Rhodopseudomonas palustris strain R1 isolated from rice field soil.

Rhodopseudomonas palustris R1 is a phototrophic, purple, non-sulfur Gram-negative bacterium first isolated from the soils of rice fields, known for its polylactic acid degradability. Here, we present the complete circular genome sequence of this bacterium, spanning 5.32 Mbp and 4,949 protein-coding sequences.

Assembly and comparative analysis of the complete mitochondrial genome of tea-scented China rose

Plant mitochondrial (mt) genomes enable better understanding of cellular processes and phylogenetic relationships. Tea-scented China rose ‘Hume's Blush Tea-scented China’ is the ancestor of the modern hybrid tea rose and has had an important and lasting influence on the breeding of the modern horticultural rose, but a comprehensive description of its mt genome is not yet available. In this study, mature leaves were used to determine the sequence of the rose mt genome. The mt genome of ‘Hume's Blush Tea-scented China’ is a circular sequence 277 730 bp in length and includes 30 protein-coding genes, 19 tRNA genes, and 3 rRNA genes. We analyzed repeat sequences, codon preferences, and RNA editing processes. In addition, we detected the transfer of 25 chloroplast genes to the mt genome, indicating intracellular genes transferred frequently from chloroplasts to mitochondria in ‘Hume's Blush Tea-scented China’. The phylogenetic analysis of the ‘Hume's Blush Tea-scented China’ mt genome and those of 26 other plant groups reflects its taxonomic status. The Ka/Ks of most genes was less than 1, indicating that most coding genes underwent negative selection, while pi was greater than 0.01, confirming highly diverse genetic variation. This work lays a foundation for future investigation of genetic variation in ‘Hume's Blush Tea-scented China’.

Closing the genome of Teredinibacter turnerae T7902 by long-read nanopore sequencing.

We present the complete closed circular genome sequence derived from Oxford Nanopore sequencing of the shipworm endosymbiont Teredinibacter turnerae T7902 (DSM 15152, ATCC 39867), originally isolated from the shipworm Lyrodus pedicellatus (1). This sequence will aid in the comparative genomics of shipworm endosymbionts and the understanding of host-symbiont evolution.

Imaging and quantification of human and viral circular RNAs.

We present a robust approach for cellular detection, imaging, localization, and quantification of human and viral encoded circular RNAs (circRNA) using amplified fluorescence in situ hybridization (ampFISH). In this procedure, a pair of hairpin probes bind next to each other at contiguous stretches of sequence and then undergo a conformational reorganization which initiates a target-dependent hybridization chain reaction (HCR) resulting in deposition of an amplified fluorescent signal at the site. By harnessing the capabilities of both ampFISH and single-molecule FISH (smFISH), we selectively identified and imaged circular RNAs and their linear counterparts derived from the human genome, SARS-CoV-2 (an RNA virus), and human cytomegalovirus (HCMV, a DNA virus). Computational image processing facilitated accurate quantification of circular RNA molecules in individual cells. The specificity of ampFISH for circular RNA detection was confirmed through an in situ RNase R treatment that selectively degrades linear RNAs without impacting circular RNAs. The effectiveness of circular RNA detection was further validated by using ampFISH probes with mismatches and probe pairs that do not bind to the continuous sequence in their target RNAs but instead bind at segregated sites. An additional specificity test involved probes against the negative strands of the circular RNA sequence, absent in the cell. Importantly, our technique allows simultaneous detection of circular RNAs and their linear counterparts within the same cell with single molecule sensitivity, enabling explorations of circular RNA biogenesis, subcellular localization, and functions.

EpiXact-ONT: Long-read whole genome sequencing for rapid outbreak detection and comprehensive plasmid transmission analysis

Background: Healthcare associated infections (HAIs) are a major contributor to patient morbidity and mortality. HAIs are increasingly important due to the rise of multidrug resistant pathogens which can lead to deadly nosocomial outbreaks. Traditional methods for investigating transmissions are slow, costly and have poor detection resolution. In addition, plasmid transmission which can horizontally transfer critical resistance and virulence genes is not part of routine infection control practice due to lack of comprehensive and cost-effective methods capable of identifying both pathogen and/or plasmid transmission. Here we demonstrate the utility of the Oxford Nanopore Technologies (ONT) platform for whole genome sequencing (WGS) based pathogen and plasmids transmission analysis. Methods: We developed a rapid end-to-end process that includes sample preparation, sequencing optimized for generating long-reads and bioinformatics workflow customized for error-prone ONT data. We use Flye to generate de novo assemblies and a secondary bioinformatics step to identify each circular sequence. Individual circular sequences with an Ori (≥1) are identified. For pathogen clonality analysis we perform a pairwise mapping-based chromosomal sequences comparison eliminating need for an external reference genome. Similarly, individual plasmids are separated and compared pairwise. We annotate both the circularized chromosomal and plasmid sequences for known resistance and virulence genes. Results: We performed ONT (and confirmatory Illumina) sequencing of the genomes of 20 bacterial isolates originating from 5 HAI investigations previously performed at Day Zero Diagnostics using epiXact®, our Illumina-based HAI sequencing and analysis lab service. ONT-based clonality determination had 100% agreement with the Illumina based pipeline. We also found that using the outbreak-specific assembled genomes instead of an external reference increased the SNP-calling resolution in the ONT pipeline. We also identified sets of clonal isolates with both identical plasmids and distinct plasmids; as well as sets of non-clonal isolates with identical plasmids and distinct plasmids. In one subset of 7 multi-species isolates, we identified 2-7 circularized plasmid sequences in each isolate, all harboring known resistance genes. 4 plasmids were found in multiple isolates, with each plasmid appearing in between 2 and 4 distinct isolates. Notably, blaNDM was identified in at least 1 plasmid in each isolate. Conclusion: We demonstrate the utility of ONT for comprehensive HAI investigations, establishing the potential to transform healthcare epidemiology with rapid outbreak determination covering pathogen and plasmid transmission in < 2 4 hours from sample receipt.

Complete genome sequence of candidate probiotic Limosilactobacillus fermentum KUFM407.

It has been reported that the administration of Limosilactobacillus fermentum alleviates diseases such as osteoporosis and colitis. In this study, we report the complete genome sequence of Limosilactobacillus fermentum KUFM407, a probiotic strain of LAB isolated from Korean traditional fermented food, Kimchi. Whole genome sequencing of L. fermentum KUFM407 was performed on the Illumina MiSeq and Oxford Nanopore MinION platform. The genome consisted of one circular chromosome (2,077,616 base pair [bp]) with a guanine cytosine (GC) content of 51.5% and one circular plasmid sequence (13,931 bp). Genome annotation identified 1,932 protein-coding genes, 15 rRNAs, and 58 tRNAs in the assembly. The function annotation of the predicted proteins revealed genes involved in the biosynthesis of bacteriocin and fatty acids. The complete genome of L. fermentum KUFM407 could provide valuable information for the development of new probiotic food and health supplements.

Full-length PacBio Amplicon Sequencing to Unveil RNA Editing Sites

Background: RNA editing enriches post-transcriptional sequence changes. Currently detecting RNA editing sites is mostly based on the Sanger sequencing platform and second-generation sequencing. However, detection with Sanger sequencing is limited by the disturbing background peaks using the direct sequencing method and the clone number using the clone sequencing method, while second-generation sequencing detection is constrained by its short read. Objective: We aimed to design a pipeline that can accurately detect RNA editing sites for full-length long-read amplicons to meet the requirement when focusing on a few specific genes of interest. Method: We developed a novel high-throughput RNA editing sites detection pipeline based on the PacBio circular consensus sequences sequencing which is accurate with high-throughput and long-read coverage. We tested the pipeline on cytosolic malate dehydrogenase in the hard-shelled mussel Mytilus coruscus and further validated it using direct Sanger sequencing. Results: Data generated from the PacBio circular consensus sequences (CCS) amplicons in three mussels were first filtered by quality and then selected by open reading frame. After filtering, 225-2047 sequences of the three mussels, respectively, were used to identify RNA editing sites. With corresponding genomic DNA sequences, we extracted 227-799 candidate RNA editing sites excluding heterozygous sites. We further figured out 7-11 final RESs using a new error model specially designed for RNA editing site detection. The resulting RNA editing sites all agree with the validation using the Sanger sequencing. Conclusion: We report a near-zero error rate method in identifying RNA editing sites of long-read amplicons with the use of PacBio CCS sequencing.

Whole genome-based antimicrobial resistance, virulence, and phylogenetic characteristics of Trueperella pyogenes clinical isolates from humans and animals

Trueperella pyogenes is an opportunistic zoonotic bacterial pathogen, whose antimicrobial resistance, virulence, and genetic relatedness between strains from animals and humans are barely studied. These characteristics were therefore analyzed for clinical T. pyogenes strains from 31 animals of 11 different species and 8 humans determining their complete circular genome sequence and antimicrobial susceptibility. The MICs of 19 antimicrobials including 3 antiseptics correlated to the resistance genes identified in silico within the genomes revealing a predominance of resistance to streptomycin (aadA9), sulfamethoxazole (sul1), and tetracycline (tet(33), tet(W/N/W)) among strains from humans and cattle. Additional resistance genes (erm(X), erm(56), cmx, drfA1, aadA1, aph(3'')-Ib (strA), aph(6)-Id (strB), aac(3)-IVa, aph(4)-Ia) were found only sporadically. The resistance genes were localized on genetic elements integrated into the chromosome. A cgMLST-based phylogenetic analysis revealed two major clusters each containing genetically diverse strains. The human strains showed the closest relatedness to strains from cattle. Virulence genes coding for fimbriae (fimA, fimC), neuroamidase (nanP, nanH), pyolysin (plo), and collagen binding protein (cbpA) were identified in strains from different hosts, but no correlation was observed between virulence factors and strain origin. The existence of resistance genes typically found in Gram-negative bacteria within the Gram-positive T. pyogenes indicates a wider capacity to adapt to antimicrobial selective pressure. Moreover, the presence of similar antimicrobial resistance profiles found in cattle and human strains as well as their closest relatedness suggests common zoonotic features and cattle as the potential source for human infections.

CircSeqAlignTk: An R package for end-to-end analysis of RNA-seq data for circular genomes

RNA sequencing (RNA-seq) technology has become one of the standard tools for studying biological mechanisms at the transcriptome level. Advances in RNA-seq technology have led to the development of numerous publicly available tools for RNA-seq data analysis. Most of these tools target linear genome sequences despite the necessity of studying organisms with circular genome sequences. For example, studying the infection mechanisms of viroids which comprise 246–401 nucleotides circular RNAs and target plants may prevent tremendous economic and agricultural damage. Unfortunately, using the available tools to construct workflows for the analysis of circular genome sequences is difficult, especially for non-bioinformaticians. To overcome this limitation, we present CircSeqAlignTk, an easy-to-use and richly documented R package. CircSeqAlignTk offers both command line and graphical user interfaces for end-to-end RNA-seq data analysis, spanning alignment to the visualisation of circular genome sequences, via a series of functions. Moreover, it includes a feature to generate synthetic sequencing data that mirrors real RNA-seq data from biological experiments. CircSeqAlignTk not only provides an easy-to-use analysis interface for novice users but also allows developers to evaluate the performance of alignment tools and new workflows.

The genome of the Arctic snow alga Limnomonas spitsbergensis (Chlamydomonadales).

Snow algae are a diverse group of extremophilic microeukaryotes found on melting polar and alpine snowfields. They play an important role in the microbial ecology of the cryosphere, and their propagation on snow and ice surfaces may in part accelerate climate-induced melting of these systems. High-quality snow algae genomes are needed for studies on their unique physiology, adaptive mechanisms, and genome evolution under multiple forms of stress, including cold temperatures and intense sunlight. Here, we assembled and annotated the genome of Limnomonas spitsbergensis, a cryophilic biciliate green alga originally isolated from melting snow on Svalbard, in the Arctic. The L. spitsbergensis genome assembly is based primarily on the use of PacBio long reads and secondly Illumina short reads, with an assembly size of 260.248 Mb in 124 contigs. A combination of 3 alternative annotation strategies was used including protein homology, RNA-seq evidence, and PacBio full-length transcript isoforms. The best merged set of annotations identified 18,277 protein-coding genes, which were 95.2% complete based on Benchmarking Universal Single-Copy Orthologs analysis. We also provide the annotated mitogenome, which is a relatively large 77.942 kb circular mapping sequence containing extensive repeats. The L. spitsbergensis genome will provide a new resource for research on snow algae adaptation, behavior, and natural selection in unique, low-temperature terrestrial environments that are under threat from climate change.

Characterization of Plasmids Isolated from Bacterial Flora of CRC Patients – Proof of Concept

Abstract Introduction Colorectal cancer (CRC) is one of the leading oncogenic disorders, both in terms of incidence and mortality. The etiology of the disease is certainly multifactorial. Various risk factors like alcohol consumption, smoking, CRC family history, inflammatory bowel disease, hormone therapy, aspirin/nonsteroidal anti-inflammatory drugs use, higher body mass index, consumption of red and/or processed meat, insufficient physical activity, and decreased intake of fruit and vegetables have been pointed out; however, there is not enough support evidence for a single particular causative mechanism. Recently, gut bacterial microbiota has been shown to influence significantly the pathogenesis of CRC. However, little attention is paid to the putative impact of plasmids in gut flora. Material and methods We have designed and tested the workflow for semi-selective isolation and amplification of random circular sequences. The exploitation of rolling circle amplification (RCA) with a random hexamers protocol is crucial for the outcome. Results Our results suggest that it is possible to isolate and amplify plasmid DNA from gut flora and further process, sequence, and identify them. Discussion Little is known about the interactions between bacterial plasmids and human cells. The collection of plasmid sequencing data and the comparison of CRC patients and healthy control sequences can be the first step to elucidating this phenomenon.

Uncovering microbiomes of the rice phyllosphere using long-read metagenomic sequencing

The plant microbiome is crucial for plant growth, yet many important questions remain, such as the identification of specific bacterial species in plants, their genetic content, and location of these genes on chromosomes or plasmids. To gain insights into the genetic makeup of the rice-phyllosphere, we perform a metagenomic analysis using long-read sequences. Here, 1.8 Gb reads are assembled into 26,067 contigs including 142 circular sequences. Within these contigs, 669 complete 16S rRNA genes are clustered into 166 bacterial species, 121 of which show low identity (<97%) to defined sequences, suggesting novel species. The circular contigs contain novel chromosomes and a megaplasmid, and most of the smaller circular contigs are defined as novel plasmids or bacteriophages. One circular contig represents the complete chromosome of a difficult-to-culture bacterium Candidatus Saccharibacteria. Our findings demonstrate the efficacy of long-read-based metagenomics for profiling microbial communities and discovering novel sequences in plant-microbiome studies.

Complete genome sequences of Lactobacillus acidophilus strain P42 and Limosilactobacillus reuteri strain P43 isolated from chicken cecum.

The gut microflora contains a diverse microbial population that is influenced by the host and the environment. We report the complete circular genome sequences of Lactobacillus acidophilus strain P42 and Limosilactobacillus reuteri strain P43 isolated from chicken cecal samples. P42 and P43 could potentially serve as poultry probiotic strains.

Genome sequence and annotation of Pyramidobacter sp. strain YE332, isolated from a cattle rumen fermentation of Leucaena leaf.

We report the 2.78-Mb circular genome sequence of Pyramidobacter sp. strain YE332, isolated from a fermentation of bovine rumen fluid, supplied with leaf material from Leucaena leucocephala cv. Cunningham. This genome sequence consists of 2,795,328 bp with 60% G + C content, 2,573 predicted coding DNA sequences, and 70 RNAs.

Complete nucleotide sequence and comparative genomic analysis of microcin B17 plasmid pMccB17.

We present a comprehensive sequence and bioinformatic analysis of the prototypical microcin plasmid, pMccb17, which includes a definitive sequence for the microcin operon, mcb. Microcin B17 (MccB17) is a ribosomally synthesized and posttranslationally modified peptide produced by Escherichia coli. It inhibits bacterial DNA gyrase similarly to quinolone antibiotics. The mcb operon, which consists of seven genes encoding biosynthetic and immunity/export functions, was originally located on the low copy number IncFII plasmid pMccB17 in the Escherichia coli strain LP17. It was later transferred to E. coli K-12 through conjugation. In this study, the plasmid was extracted from the E. coli K-12 strain RYC1000 [pMccB17] and sequenced twice using an Illumina short-read method. The first sequencing was conducted with the host bacterial chromosome, and the plasmid DNA was then purified and sequenced separately. After assembly into a single contig, polymerase chain reaction primers were designed to close the single remaining gap via Sanger sequencing. The resulting complete circular DNA sequence is 69,190 bp long and includes 81 predicted genes. These genes were initially identified by Prokka and subsequently manually reannotated using BLAST. The plasmid was assigned to the F2:A-:B- replicon type with a MOBF12 group conjugation system. A comparison with other IncFII plasmids revealed a large proportion of shared genes, particularly in the conjugative plasmid backbone. However, unlike many contemporary IncFII plasmids, pMccB17 lacks transposable elements and antibiotic resistance genes. In addition to the mcb operon, this plasmid carries 25 genes of unknown function.

De novo genome assembly and pan-genome analysis of the fast-growing Indian isolates of Synechococcus elongatus: Potential chassis for bioproduction

Cyanobacteria are explored as phototrophic cell factories for the conversion of CO2 to chemicals. Although several cyanobacterial platforms have been established, discovering novel cyanobacterial strains with different growth and metabolic traits would be advantageous. Here, we describe de novo genome assembly and pan-genome analysis of eight fast-growing Indian isolates of Synechococcus elongatus, displaying an average doubling time of 3–4 h. The draft genome sequences of S. elongatus PCC 11801 and PCC 11802 were earlier reported by our group. However, here we present circular un-gapped genome sequences of all the eight strains. Their pan-genome analysis showed that these strains form a distinct sub-clade within the S. elongatus clade, and are closely related to a widely used cyanobacterium S. elongatus PCC 7942. Pan-genome and extra-chromosomal DNA sequence analyses further revealed intriguing features, including CRISPR-associated protein genes, circular episomal elements, and other essential genes which were previously not reported for this clade. Further, from bio-engineering perspective, we optimized their transformation using neutral site integrative plasmids. The heterologous protein expression was confirmed using eYFP reporter system, and validated by engineering a mannitol biosynthesis pathway in these strains. Although the strains exhibited comparable eYFP fluorescence, the mannitol production varied significantly, indicating physiological differences among the strains. The maximum mannitol was obtained from S. elongatus IITB6 strain. Overall, this work encompasses comparative growth analysis of the eight Indian isolates of S. elongatus, their genome sequences and pan-genome analysis. We demonstrated their potential as phototrophic cell factories, providing cyanobacterial bio-chassis options.

Enhancing transcriptome analysis in medicinal plants: multiple unigene sets in Astragalus membranaceus.

Astragalus membranaceus is a medicinal plant mainly used in East Asia and contains abundant secondary metabolites. Despite the importance of this plant, the available genomic and genetic information is still limited. De novo transcriptome construction is recognized as an essential method for transcriptome research when reference genome information is incomplete. In this study, we constructed three individual transcriptome sets (unigene sets) for detailed analysis of the phenylpropanoid biosynthesis pathway, a major metabolite of A. membranaceus. Set-1 was a circular consensus sequence (CCS) generated using PacBio sequencing (PacBio-seq). Set-2 consisted of hybridized assembled unigenes with Illumina sequencing (Illumina-seq) reads and PacBio CCS using rnaSPAdes. Set-3 unigenes were assembled from Illumina-seq reads using the Trinity software. Construction of multiple unigene sets provides several advantages for transcriptome analysis. First, it provides an appropriate expression filtering threshold for assembly-based unigenes: a threshold transcripts per million (TPM) ≥ 5 removed more than 88% of assembly-based unigenes, which were mostly short and low-expressing unigenes. Second, assembly-based unigenes compensated for the incomplete length of PacBio CCSs: the ends of the 5`/3` untranslated regions of phenylpropanoid-related unigenes derived from set-1 were incomplete, which suggests that PacBio CCSs are unlikely to be full-length transcripts. Third, more isoform unigenes could be obtained from multiple unigene sets; isoform unigenes missing in Set-1 were detected in set-2 and set-3. Finally, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that phenylpropanoid biosynthesis and carbohydrate metabolism were highly activated in A. membranaceus roots. Various sequencing technologies and assemblers have been developed for de novo transcriptome analysis. However, no technique is perfect for de novo transcriptome analysis, suggesting the need to construct multiple unigene sets. This method enables efficient transcript filtering and detection of longer and more diverse transcripts.

CirCodAn: A GHMM-based tool for accurate prediction of coding regions in circRNA.

Studies focusing on characterizing circRNAs with the potential to translate into peptides are quickly advancing. It is helping to elucidate the roles played by circRNAs in several biological processes, especially in the emergence and development of diseases. While various tools are accessible for predicting coding regions within linear sequences, none have demonstrated accurate open reading frame detection in circular sequences, such as circRNAs. Here, we present cirCodAn, a novel tool designed to predict coding regions in circRNAs. We evaluated the performance of cirCodAn using datasets of circRNAs with strong translation evidence and showed that cirCodAn outperformed the other tools available to perform a similar task. Our findings demonstrate the applicability of cirCodAn to identify coding regions in circRNAs, which reveals the potential of use of cirCodAn in future research focusing on elucidating the biological roles of circRNAs and their encoded proteins. cirCodAn is freely available at https://github.com/denilsonfbar/cirCodAn.

Screening and Characterization of Functional circRNAs in Neuronal Cultures.

This chapter describes a methodology for the screening and characterization of functional circRNAs, particularly in the context of neural circuit development. Taking advantage of a primary rat neuron culture model of synaptogenesis, we propose a means of selecting from the plethora of circRNA species based on their expression levels, dendritic localization, conservation, and activity regulation. These candidates are then knocked down with RNAi approaches in a functional screen for their potential role in the formation and maturation of excitatory synapses.Upon identification of top candidates regulating synaptogenesis, we tie together different "Omics" approaches to explore the molecular mechanisms underlying the phenotypes observed upon circRNA knockdown. We utilized our EnrichMir algorithm to identify overrepresented miRNA binding sites in differentially expressed genes from polyA-RNA-seq following circRNA knockdown. Furthermore, our ScanMiR web tool allows for the miRNA binding prediction of reconstructed internal circular RNA sequences. Small-RNA sequencing is used to monitor changes in miRNA levels in the circRNA knockdown to complement results obtained from EnrichMiR. Finally, the experimental validation of promising miRNA-circRNA pairs sets the stage for in-depth biochemical exploration of the circRNA interactome and mechanism of action.