Benchmarking Long-Read Assemblers for Genomic Analyses of Bacterial Pathogens Using Oxford Nanopore Sequencing

Oxford Nanopore sequencing can be used to achieve complete bacterial genomes. However, the error rates of Oxford Nanopore long reads are greater compared to Illumina short reads. Long-read assemblers using a variety of assembly algorithms have been developed to overcome this deficiency, which have not been benchmarked for genomic analyses of bacterial pathogens using Oxford Nanopore long reads. In this study, long-read assemblers, namely Canu, Flye, Miniasm/Racon, Raven, Redbean, and Shasta, were thus benchmarked using Oxford Nanopore long reads of bacterial pathogens. Ten species were tested for mediocre- and low-quality simulated reads, and 10 species were tested for real reads. Raven was the most robust assembler, obtaining complete and accurate genomes. All Miniasm/Racon and Raven assemblies of mediocre-quality reads provided accurate antimicrobial resistance (AMR) profiles, while the Raven assembly of Klebsiella variicola with low-quality reads was the only assembly with an accurate AMR profile among all assemblers and species. All assemblers functioned well for predicting virulence genes using mediocre-quality and real reads, whereas only the Raven assemblies of low-quality reads had accurate numbers of virulence genes. Regarding multilocus sequence typing (MLST), Miniasm/Racon was the most effective assembler for mediocre-quality reads, while only the Raven assemblies of Escherichia coli O157:H7 and K. variicola with low-quality reads showed positive MLST results. Miniasm/Racon and Raven were the best performers for MLST using real reads. The Miniasm/Racon and Raven assemblies showed accurate phylogenetic inference. For the pan-genome analyses, Raven was the strongest assembler for simulated reads, whereas Miniasm/Racon and Raven performed the best for real reads. Overall, the most robust and accurate assembler was Raven, closely followed by Miniasm/Racon.     

De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora)

Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora.     

Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing

Small open reading frames (sORFs) have translational potential to produce peptides that play essential roles in various biological processes. Nevertheless, many sORF-encoded peptides (SEPs) are still on the prediction level. Here, we construct a strategy to analyze SEPs by combining top-down and de novo sequencing to improve SEP identification and sequence coverage. With de novo sequencing, we identified 1682 peptides mapping to 2544 human sORFs, which were all first characterized in this work. Two-thirds of these new sORFs have reading frame shifts and use a non-ATG start codon. The top-down approach identified 241 human SEPs, with high sequence coverage. The average length of the peptides from the bottom-up database search was 19 amino acids (AA); from de novo sequencing, it was 9 AA; and from the top-down approach, it was 25 AA. The longer peptide positively boosts the sequence coverage, more efficiently distinguishing SEPs from the known gene coding sequence. Top-down has the advantage of identifying peptides with sequential K/R or high K/R content, which is unfavorable in the bottom-up approach. Our method can explore new coding sORFs and obtain highly accurate sequences of their SEPs, which can also benefit future function research.     

Whole Genome Sequencing,Focused Assays and Functional Studies Increasing Understanding in Cryptic Inherited Retinal Dystrophies

The inherited retinal dystrophies (IRDs) are a clinically and genetically complex group of disorders primarily affecting the rod and cone photoreceptors or other retinal neuronal layers, with emerging therapies heralding the need for accurate molecular diagnosis. Targeted capture and panel-based strategies examining the partial or full exome deliver molecular diagnoses in many IRD families tested. However, approximately one in three families remain unsolved and unable to obtain personalised recurrence risk or access to new clinical trials or therapy. In this study, we investigated whole genome sequencing (WGS), focused assays and functional studies to assist with unsolved IRD cases and facilitate integration of these approaches to a broad molecular diagnostic clinical service. The WGS approach identified variants not covered or underinvestigated by targeted capture panel-based clinical testing strategies in six families. This included structural variants, with notable benefit of the WGS approach in repetitive regions demonstrated by a family with a hybrid gene and hemizygous missense variant involving the opsin genes, OPN1LW and OPN1MW. There was also benefit in investigation of the repetitive GC-rich ORF15 region of RPGR. Further molecular investigations were facilitated by focused assays in these regions. Deep intronic variants were identified in IQCB1 and ABCA4, with functional RNA based studies of the IQCB1 variant revealing activation of a cryptic splice acceptor site. While targeted capture panel-based methods are successful in achieving an efficient molecular diagnosis in a proportion of cases, this study highlights the additional benefit and clinical value that may be derived from WGS, focused assays and functional genomics in the highly heterogeneous IRDs.     

Comparison of Long-Read Methods for Sequencing and Assembly of Lepidopteran Pest Genomes

Lepidopteran species are mostly pests, causing serious annual economic losses. High-quality genome sequencing and assembly uncover the genetic foundation of pest occurrence and provide guidance for pest control measures. Long-read sequencing technology and assembly algorithm advances have improved the ability to timeously produce high-quality genomes. Lepidoptera includes a wide variety of insects with high genetic diversity and heterozygosity. Therefore, the selection of an appropriate sequencing and assembly strategy to obtain high-quality genomic information is urgently needed. This research used silkworm as a model to test genome sequencing and assembly through high-coverage datasets by de novo assemblies. We report the first nearly complete telomere-to-telomere reference genome of silkworm Bombyx mori (P50T strain) produced by Pacific Biosciences (PacBio) HiFi sequencing, and highly contiguous and complete genome assemblies of two other silkworm strains by Oxford Nanopore Technologies (ONT) or PacBio continuous long-reads (CLR) that were unrepresented in the public database. Assembly quality was evaluated by use of BUSCO, Inspector, and EagleC. It is necessary to choose an appropriate assembler for draft genome construction, especially for low-depth datasets. For PacBio CLR and ONT sequencing, NextDenovo is superior. For PacBio HiFi sequencing, hifiasm is better. Quality assessment is essential for genome assembly and can provide better and more accurate results. For chromosome-level high-quality genome construction, we recommend using 3D-DNA with EagleC evaluation. Our study references how to obtain and evaluate high-quality genome assemblies, and is a resource for biological control, comparative genomics, and evolutionary studies of Lepidopteran pests and related species.     

SBR工艺除磷过程与种群结构在线监测

For efficient energy consumption and control of effluent quality, the cycle duration for a sequencing batch reactor (SBR) needs to be adjusted by real-time control according to the characteristics and loading of wastewater. In this study, an on-line information system for phosphorus removal processes was established. Based on the analysis for four systems with different ecological community structures and two operation modes, anaerobic-aerobic process and anaerobic-anaerobic process, the characteristic patterns of oxidation-reduction potential (ORP) and pH were related to phosphorous dynamics in the anaerobic, anoxic and aerobic phases, for determination of the end of phosphorous removal. In the operation mode of anaerobic-aerobic process, the pH profile in the anaerobic phase was used to estimate the relative amount of phosphorous accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), which is beneficial to early detection of ecology community shifts. The on-line sensor values of pH and ORP may be used as the parameters to adjust the duration for phosphorous removal and community shifts to cope with influent variations and maintain appropriate operation conditions.     

CRISPR/Cas9–Mediated Genome Editing for Pseudomonas fulva,a Novel Pseudomonas Species with Clinical,Animal, and Plant–Associated Isolates

As one of the most widespread groups of Gram–negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant–associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time–consuming and laborious endeavor. Here, by using the CRISPR–Cas system, a versatile gene–editing tool with a two–plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic–related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked–in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species.     

Integrating the Roles for Cytokinin and Auxin in De Novo Shoot Organogenesis: From Hormone Uptake to Signaling Outputs

De novo shoot organogenesis (DNSO) is a procedure commonly used for the in vitro regeneration of shoots from a variety of plant tissues. Shoot regeneration occurs on nutrient media supplemented with the plant hormones cytokinin (CK) and auxin, which play essential roles in this process, and genes involved in their signaling cascades act as master regulators of the different phases of shoot regeneration. In the last 20 years, the genetic regulation of DNSO has been characterized in detail. However, as of today, the CK and auxin signaling events associated with shoot regeneration are often interpreted as a consequence of these hormones simply being present in the regeneration media, whereas the roles for their prior uptake and transport into the cultivated plant tissues are generally overlooked. Additionally, sucrose, commonly added to the regeneration media as a carbon source, plays a signaling role and has been recently shown to interact with CK and auxin and to affect the efficiency of shoot regeneration. In this review, we provide an integrative interpretation of the roles for CK and auxin in the process of DNSO, adding emphasis on their uptake from the regeneration media and their interaction with sucrose present in the media to their complex signaling outputs that mediate shoot regeneration.     

Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses

Some prevention strategies, including vaccines and antibiotic alternatives, have been developed to reduce enterotoxigenic Escherichia coli proliferation in animal production. In this study, a wild-type strain of BE311 with a virulent heat-stable enterotoxin gene identical to E. coli K99 was isolated for its high potential for gene expression ability. The whole genome of E. coli BE311 was sequenced for gene analyses and editing. Subsequently, the fluorescent gene mCherry was successfully knocked into the genome of E. coli BE311 by CRISPR/Cas9. The E. coli BE311–mCherry strain was precisely quantified through the fluorescence intensity and red colony counting. The inflammatory factors in different intestinal tissues all increased significantly after an E. coli BE311–mCherry challenge in Sprague–Dawley rats (p < 0.05). The heat-stable enterotoxin gene of E. coli BE311 was knocked out, and an attenuated vaccine host E. coli BE311-ST^KO was constructed. Flow cytometry showed apoptotic cell numbers were lower following a challenge of IPEC-J2 cells with E. coli BE311-ST^KO than with E. coli BE311. Therefore, the E. coli BE311–mCherry and E. coli BE311-ST^KO strains that were successfully constructed based on the gene knock-in and knock-out technology could be used as ideal candidates in ETEC challenge models and for the development of attenuated vaccines.     

An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and Single-Cell RNA Sequencing

The recent advent of single-cell RNA sequencing (scRNA-seq) has enabled access to the developmental landscape of a complex organ by monitoring the differentiation trajectory of every specialized cell type at the single-cell level. A main challenge in this endeavor is dissociating plant cells from the rigid cell walls and some species are recalcitrant to such cellular isolation. Here, we describe the establishment of a simple and efficient protocol for protoplast preparation in Chirita pumila, which includes two consecutive digestion processes with different enzymatic buffers. Using this protocol, we generated viable cell suspensions suitable for an array of expression analyses, including scRNA-seq. The universal application of this protocol was further tested by successfully isolating high-quality protoplasts from multiple organs (petals, fruits, tuberous roots, and gynophores) from representative species on the key branches of the angiosperm lineage. This work provides a robust method in plant science, overcoming barriers to isolating protoplasts in diverse plant species and opens a new avenue to study cell type specification, tissue function, and organ diversification in plants.     

Candidate Genes in Testing Strategies for Linkage Analysis and Bioinformatic Sorting of Whole Genome Sequencing Data in Three Small Japanese Families with Idiopathic Superior Oblique Muscle Palsy

Idiopathic superior oblique muscle palsy is a major type of paralytic, non-comitant strabismus and presents vertical and cyclo-torsional deviation of one eye against the other eye, with a large vertical fusion range and abnormal head posture such as head tilt. Genetic background is considered to play a role in its development, as patients with idiopathic superior oblique muscle palsy have varying degrees of muscle hypoplasia and, rarely, the complete absence of the muscle, that is, aplasia. In this study, whole genome sequencing was performed, and single nucleotide variations and short insertions/deletions (SNVs/InDels) were annotated in two patients each in three small families (six patients in total) with idiopathic superior oblique muscle palsy, in addition to three normal individuals in one family. At first, linkage analysis was carried out in the three families and SNVs/InDels in chromosomal loci with negative LOD scores were excluded. Next, SNVs/InDels shared by the six patients, but not by the three normal individuals, were chosen. SNVs/InDels were further narrowed down by choosing low-frequency (<1%) or non-registered SNVs/InDels in four databases for the Japanese population, and then by choosing SNVs/InDels with functional influence, leading to one candidate gene, SSTR5-AS1 in chromosome 16. The six patients were heterozygous for 13-nucleotide deletion in SSTR5-AS1, except for one homozygous patient, while the three normal individuals were wild type. Targeted polymerase chain reaction (PCR) and direct sequencing of PCR products confirmed the 13-nucleotide deletion in SSTR5-AS1. In the face of newly-registered SSTR5-AS1 13-nucleotide deletion at a higher frequency in a latest released database for the Japanese population, the skipping of low-frequency and non-registration sorting still resulted in only 13 candidate genes including SSTR5-AS1 as common variants. The skipping of linkage analysis also led to the same set of 13 candidate genes. Different testing strategies that consisted of linkage analysis and simple unintentional bioinformatics could reach candidate genes in three small families with idiopathic superior oblique muscle palsy.