Large Insertion Research Articles

Long-read sequencing enables comprehensive molecular genetic diagnosis of Fabry disease

BackgroundThe clinical diagnosis of Fabry Disease (FD) can be challenging due to the clinical heterogeneity, especially in females. Patients with FD often experience a prolonged interval between the onset of symptoms and receiving a diagnosis. Genetic testing is the gold standard for precise diagnosis of FD, however conventional genetic testing could miss deep intronic variants and large deletions or duplications. Although next-generation sequencing, which analyzes numerous genes, has been successfully used for FD diagnosis and can detect complex variants, an effective and rapid tool for identifying a wide range of variants is imminent, contributing to decrease the diagnostic delay.MethodsThe comprehensive Analysis of FD (CAFD) assay was developed for FD genetic diagnosis, employing long-range PCR coupled with long-read sequencing to target the full-length GLA gene and its flanking regions. Its clinical performance was assessed through a comparative analysis with Sanger sequencing.ResultsGenetic testing was performed on 82 individuals, including 48 probands and 34 relatives. The CAFD assay additionally identified variants in two probands: one had a novel and de novo pathogenic variant with a 1715 bp insertion in intron 4, and the other carried two deep intronic VUS variants in cis-configuration also in intron 4. In total, CAFD identified 47 different variants among 48 probands. Of these, 42 (89.36%, 42/47) were pathogenic, while 5 (10.64%, 5/47) were VUS. Sixteen (34.04%, 16/47) of the variants were novel, including 15 SNV/Indels and one large intronic insertion. Pedigree analysis of 21 probands identified four de novo disease-causing variants. Hence, FD exhibits not only variable clinical presentations but also a wide spectrum of variants. Utilizing a comprehensive testing algorithm for diagnosing FD, which includes enzyme activity, clinical features, and genetic testing, the diagnostic yield of CAFD is 97.92% (47/48), which is higher than that of conventional Sanger sequencing, at 95.83% (46/48).ConclusionThe duration between initial clinical presentation and diagnosis remains long and winding. CAFD provides precise diagnosis for a wide spectrum of GLA variants, promoting timely diagnosis and appropriate treatment for FD patients.

Molecular Biodiversity in Fusarium subglutinans and F. temperatum: A Valuable Tool to Distinguish the Two Sister Species and Determine the Beauvericin Chemotype.

Fusarium subglutinans and F. temperatum are widely distributed maize pathogens recognized as distinct species with a species-specific chemotype based on patterns of mycotoxins. Recent comparative genomic analysis revealed that genomes of both species carry a complete beauvericin (Bea) biosynthetic genes cluster, but the key gene Bea1 in F. subglutinans is not functional likely due to a large insertion (NRPS22ins) and multiple mutations (SNP298 and SNP528). We used the recently published genome sequences for these species to develop PCR markers for investigating the distribution of three main mutations in the Bea1 gene in a large collection of strains of both species from around the world. We also designed a PCR assay for a rapid and reliable discrimination of both species in the evaluation of crop exposure to mycotoxins. Overall, our results showed that SNP528 was the most common mutation, followed by NRPS22ins and SNP298. Moreover, phylogenetic analyses suggest that non-synonymous SNPs have occurred first, and that the resulting inactivation of BEA production has caused the accumulation of other polymorphisms, including the NRPS22ins, in the entire gene-coding region. The screening for genetic differences between these species could guide future crop management strategies.

Enhancing SARS-CoV-2 Lineage Surveillance through the Integration of a Simple and Direct qPCR-Based Protocol Adaptation with Established Machine Learning Algorithms.

Emerging and evolving Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) lineages, adapted to changing epidemiological conditions, present unprecedented challenges to global public health systems. Here, we introduce an adapted analytical approach that complements genomic sequencing, applying a cost-effective quantitative polymerase chain reaction (qPCR)-based assay. Viral RNA samples from SARS-CoV-2 positive cases detected by diagnostic laboratories or public health network units in Ceará, Brazil, were tracked for genomic surveillance and analyzed by using paired-end sequencing combined with integrative genomic analysis. Validation of a key structural variation was conducted with gel electrophoresis for the presence of a specific open reading frame 7a(ORF7a) gene deletion within the "BE.9" lineages tracked. The analytical innovation of our method is the optimization of a simple intercalating dye-based qPCR assay through repositioning primers from the ARTIC v4.1 amplicon panel to detect large molecular patterns. This assay distinguishes between "BE.9" and "non-BE.9" lineages, particularly BQ.1, without the need for expensive probes or sequencing. The protocol was validated against lineage predictions from next-generation sequencing (NGS) using 525 paired samples, achieving 93.3% sensitivity, 95.1% specificity, and 92.4% agreement, as measured by Cohen's Kappa coefficient. Machine learning (ML) models were trained using the melting curves from intercalating dye-based qPCR of 1724 samples, enabling highly accurate lineage assignment. Among them, the support vector machine (SVM) model had the best performance and after fine-tuning showed ∼96.52% (333/345) accuracy in comparison to the test data set. Our integrated approach provides an adapted analytical method that is both cost-effective and scalable, suitable for rapid assessment of emerging variants, especially in resource-limited settings. In this work, the protocol is applied to improve the monitoring of SARS-CoV-2 sublineages but can be extended to track any key molecular signature, including large insertions and deletions (indels) commonly observed in pathogenic agent subtypes. By offering a complement to traditional sequencing methods and utilizing easily trainable machine learning algorithms, our methodology contributes to enhanced molecular surveillance strategies and supports global efforts in pandemic control.

A MYB transcription factor underlying plant height in sorghum qHT7.1 and maize Brachytic 1 loci.

Manipulating plant height is an essential component of crop improvement. Plant height was generally reduced through breeding in wheat, rice, and sorghum to resist lodging and increase grain yield but kept high for bioenergy crops. Here, we positionally cloned a plant height quantitative trait locus (QTL) qHT7.1 as a MYB transcription factor controlling internode elongation, cell proliferation, and cell morphology in sorghum. A 740 bp transposable element insertion in the intronic region caused a partial mis-splicing event, generating a novel transcript that included an additional exon and a premature stop codon, leading to short plant height. The dominant allele had an overall higher expression than the recessive allele across development and internode position, while both alleles' expressions peaked at 46 days after planting and progressively decreased from the top to lower internodes. The orthologue of qHT7.1 was identified to underlie the brachytic1 (br1) locus in maize. A large insertion in exon 3 and a 160 bp insertion at the promoter region were identified in the br1 mutant, while an 18 bp promoter insertion was found to be associated with reduced plant height in a natural recessive allele. CRISPR/Cas9-induced gene knockout of br1 in two maize inbred lines showed significant plant height reduction. These findings revealed functional connections across natural, mutant, and edited alleles of this MYB transcription factor in sorghum and maize. This enriched our understanding of plant height regulation and enhanced our toolbox for fine-tuning plant height for crop improvement.

Comprehensive analysis of Mycobacterium tuberculosis genomes reveals genetic variations in bacterial virulence

Tuberculosis, a disease caused by Mycobacterium tuberculosis (Mtb), is a significant health problem worldwide. Here, we developed a method to detect large insertions and deletions (indels), which have been generally understudied. Leveraging this framework, we performed a comprehensive analysis of single nucleotide variants and small and large indels across 1,960 Mtb clinical isolates. Comparing the distribution of variants demonstrated that gene disruptive variants are underrepresented in genes essential for bacterial survival. An evolutionary analysis revealed that Mtb genomes are enriched in partially deleterious mutations. Genome-wide association studies identified small and large deletions in eccB2 significantly associated with patient prognosis. Additionally, we unveil significant associations with antibiotic resistance in 23 non-canonical genes. Among these, large indels are primarily found in genetic regions of Rv1216c, Rv1217c, fadD11, and ctpD. This study provides a comprehensive catalog of genetic variations and highlights their potential impact for the future treatment and risk prediction of tuberculosis.

Diversification of Antibodies: From V(D)J Recombination to Somatic Exon Shuffling.

Antibodies that gain specificity by a large insert encoding for an extra domain were described for the first time in 2016. In malaria-exposed individuals, an exon deriving from the leukocyte-associated immunoglobulin-like 1 (LAIR1) gene integrated via a copy-and-paste insertion into the immunoglobulin heavy chain encoding region. A few years later, a second example was identified, namely a dual exon integration from the leukocyte immunoglobulin-like receptor B1 (LILRB1) gene that is located in close proximity to LAIR1. A dedicated high-throughput characterization of chimeric immunoglobulin heavy chain transcripts unraveled, that insertions from distant genomic regions (including mitochondrial DNA) can contribute to human antibody diversity. This review describes the modalities of insert-containing antibodies. The role of known DNA mobility aspects, such as genomic translocation, gene conversion, and DNA fragility, is discussed in the context of insert-antibody generation. Finally, the review covers why insert antibodies were omitted from the past repertoire analyses and how insert antibodies can contribute to protective immunity or an autoreactive response.

Evaluation of the Polypyrrole Coupling Mode for High-Performance Dual-Ion Batteries.

Due to the advantages of large interstitial sites, antisolubility, and reversible insertion and extraction of anions, polypyrrole (PPy) has become an excellent P-type electrode material for dual-ion batteries. Unfortunately, PPy electrodes inevitably suffer from low specific capacity and poor cycle stability because of structural disintegration during repeated cycling as well as poor doping ability brought on by aggregation or cross-linking within the PPy chain. In this work, PPy with different proportions of coupling mode (α-α, α-β, or β-β coupling) was derived from different preparation methods. Among them, PPy derived from the interfacial frozen polymerization method (I-PPy) is dominated by the α-α coupling mode and possesses the best anion doping ability and the highest specific capacity of 119 mAh g-1 at 100 mA g-1 compared with PPy derived from electrochemical deposition (E-PPy) and chemical oxidation method (C-PPy) (both less than 40 mAh g-1). This work verifies that increasing the proportion of the α-α coupling mode in the PPy electrode is a useful strategy to enhance the anion doping ability and capacity of dual-ion batteries.

Differential contributions of double-strand break repair pathways to DNA rearrangements following the irradiation of Arabidopsis seeds and seedlings with ion beams.

DNA rearrangements, including inversions, translocations, and large insertions/deletions (indels), are crucial for crop evolution, domestication, and improvement. The rearrangements are frequently induced by ion beams via the mis-repair of DNA double-strand breaks (DSBs). Unfortunately, how ion beam-induced DSBs are repaired has not been comprehensively analyzed and the mechanisms underlying DNA rearrangements remain unclear. In this study, clonal sectors originating from single mutated cells in carbon ion-irradiated plants were used for whole-genome sequencing analyses after Arabidopsis seeds and seedlings were irradiated. Comparative analyses of the induced mutations (e.g., size and frequency of indels and microhomology at the junctions of the rearrangements) in the irradiated materials suggested that the broken/rejoined DSB ends were more extensively processed in seedlings than in seeds. A mutation to canonical non-homologous end-joining (c-NHEJ), which is a DSB repair pathway with minimal processing of DSB ends, increased the sensitivity to ion beams more in the seeds than in the seedlings, which was consistent with the junction analysis results, indicative of the minor contribution of c-NHEJ to the carbon ion-induced DSB repair in seedlings. Considering the characteristics of the large templated insertions in irradiated seedlings, ion-beam-induced DSBs in seedlings are likely repaired primarily by a polymerase theta-mediated pathway. Polymerase theta-deficient seedlings were more sensitive to ion beams than the c-NHEJ-deficient seedlings, consistent with this hypothesis. This study revealed the key characteristics of ion beam-induced DSBs and the associated repair mechanisms related to the physiological status of the irradiated materials, with implications for elucidating the occurrence and induction of rearrangements.

Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.

Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing outcomes. We recently leveraged a type I-F CRISPR-associated transposase (CAST) from the Pseudoalteromonas Tn7016 transposon (PseCAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. PseCAST is the only characterized CAST system that has achieved human genomic DNA insertions, but multiple lines of evidence suggest that DNA binding may be a critical bottleneck that limits high-efficiency activity. Here we report structural determinants of target DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), which revealed novel subtype-specific interactions and RNA-DNA heteroduplex features. By combining our structural data with target DNA library screens and rationally engineered protein mutations, we uncovered CAST variants that exhibit increased integration efficiency and modified PAM stringency. Structure predictions of key interfaces in the transpososome holoenzyme also revealed opportunities for the design of hybrid CASTs, which we leveraged to build chimeric systems that combine high-activity DNA binding and DNA integration modules. Collectively, our work provides unique structural insights into type I-F CAST systems while showcasing multiple diverse strategies to investigate and engineer new RNA-guided transposase architectures for human genome editing applications.

Cytoplasmic genomes of Jasminum sambac reveal divergent sub-mitogenomic conformations and a large nuclear chloroplast-derived insertion

BackgroundJasminum sambac, a widely recognized ornamental plant prized for its aromatic blossoms, exhibits three flora phenotypes: single-petal (“SP”), double-petal (“DP”), and multi-petal (“MP”). The lack of detailed characterization and comparison of J. sambac mitochondrial genomes (mitogenomes) hinders the exploration of the genetic and structural diversity underlying the varying floral phenotypes in jasmine accessions.ResultsHere, we de novo assembled three mitogenomes of typical phenotypes of J. sambac, “SP”, “DP”, and “MP-hutou” (“HT”), with PacBio reads and the “HT” chloroplast (cp) genome with Illumina reads, and verified them with read mapping and fluorescence in situ hybridization (FISH). The three mitogenomes present divergent sub-genomic conformations, with two, two, and four autonomous circular chromosomes ranging in size from 35.7 kb to 405.3 kb. Each mitogenome contained 58 unique genes. Ribosome binding sites with conserved AAGAAx/AxAAAG motifs were detected upstream of uncanonical start codons TTG, CTG and GTG. The three mitogenomes were similar in genomic content but divergent in structure. The structural variations were mainly attributed to recombination mediated by a large (~ 5 kb) forward repeat pair and several short repeats. The three jasmine cp. genomes showed a well-conserved structure, apart from a 19.9 kb inversion in “HT”. We identified a 14.3 kb “HT”-specific insertion on Chr7 of the “HT” nuclear genome, consisting of two 7 kb chloroplast-derived fragments with two intact ndhH and rps15 genes, further validated by polymerase chain reaction (PCR). The well-resolved phylogeny suggests faster mitogenome evolution in J. sambac compared to other Oleaceae species and outlines the mitogenome evolutionary trajectories within Lamiales. All evidence supports that “DP” and “HT” evolved from “SP”, with “HT” being the most recent derivative of “DP”.ConclusionThe comprehensive characterization of jasmine organelle genomes has added to our knowledge of the structural diversity and evolutionary trajectories behind varying jasmine traits, paving the way for in-depth exploration of mechanisms and targeted genetic research.

Chromosome breakage-replication/fusion enables rapid DNA amplification.

DNA rearrangements are thought to arise from two classes of processes. The first class involves DNA breakage and fusion ("cut-and-paste") without net DNA gain or loss. The second class involves aberrant DNA replication ("copy-and-paste") and can produce either net DNA gain or loss. We previously demonstrated that the partitioning of chromosomes into aberrant structures of the nucleus, micronuclei or chromosome bridges, can generate cut-and-paste rearrangements by chromosome fragmentation and ligation. Surprisingly, in the progeny clones of single cells that have undergone chromosome bridge breakage, we identified large segmental duplications and short sequence insertions that are commonly attributed to copy-and-paste processes. Here, we demonstrate that both large duplications and short insertions are inherent outcomes of the replication and fusion of unligated DNA ends, a process we term breakage-replication/fusion (B-R/F). We propose that B-R/F provides a unifying explanation for complex rearrangement patterns including chromothripsis and chromoanasynthesis and enables rapid DNA amplification after chromosome fragmentation.

MAX germline mutation-associated pheochromocytoma-paraganglioma syndrome: multiple endocrine neoplasia type 5

The individualized precision management of hereditary pheochromocytoma (PHEO) and paraganglioma (PGL) syndromes (PPGLs) based on molecular diagnosis and molecular subtype is becoming more popular. The newly discovered MAX germline mutation-associated PPGLs are autosomally dominant and rare. To raise awareness and explore the effective management of individual diagnosis and treatment, the relevant literature published between January 2011 and February was systematically reviewed. There were a total of 101 patients in the 77 families, involving all 5 exons, containing 44 types of MAX germline mutations and mostly concentrated in exons 3 and 4 (64.4%), the main mutations were nonsense mutations and missense mutations (72.3%), and some were large fragment deletions or insertions, intron variant, gene fusion mutations were relatively infrequent. Furthermore, about 10% of the patients had a paternal parent-of-origin effect. Among the 101 patients, 96 (95.0%) developed PHEO including 15 metastatic PHEO, 61 bilateral PHEO and 35 unilateral PHEO. The age of diagnosis was (31.7±10.9) years (range: 13 to 80 years). The male to female ratio was 1.2∶1. Eleven were accompanied with chest and abdominal PGL. Eight (7.9%) were accompanied by functional pituitary adenoma. And 12 (11.9%) developed other neuroendocrine tumors (NET), of which 8 were accompanied by PHEO, including 4 hyperparathyroidism, 1 gangliocytoma and neuroblastoma, 1 pancreatic NET, 1 medullary thyroid carcinoma and 1 C cell hyperplasia. Six presented concomitant non-NET, including 1 tongue squamous cell carcinoma, 1 papillary thyroid carcinoma, 1 prostate cancer, 1 renal oncocytoma, 1 breast cancer with renal oncocytoma, and 1 thoracic chondrosarcoma with multifocal adenocarcinoma of lung. The remaining 5 cases (5.0%), including 4 other NET (2 ganglioblastoma, 1 abdominal neuroblastoma and 1 pancreatic NET) and 1 asymptomatic child, did not present PHEO. The MAX germline mutation may cause a novel multiple endocrine neoplasia, which can be described as type 5. A comprehensive baseline assessment of neural crest cell-derived diseases such as PPGL, pituitary adenoma, hyperparathyroidism, and/or gangliocytoma (neuroblastoma) was recommended for all people with MAX germline mutations, and the risk of bilateral and/or metastatic PHEO should also be considered. In contrast, patients with PPGLs combined with other NET, such as functional pituitary adenoma, should undergo genetic testing and pedigree screening that includes at least the MAX gene.

Construction and Characterization of a High-Capacity Replication-Competent Murine Cytomegalovirus Vector for Gene Delivery.

We investigated the basic characteristics of a new murine cytomegalovirus (MCMV) vector platform. Using BAC technology, we engineered replication-competent recombinant MCMVs with deletions of up to 26% of the wild-type genome. To this end, we targeted five gene blocks (m01-m17, m106-m109, m129-m141, m144-m158, and m159-m170). BACs featuring deletions from 18% to 26% of the wild-type genome exhibited delayed virus reconstitution, while smaller deletions (up to 16%) demonstrated reconstitution kinetics similar to those of the wild type. Utilizing an innovative methodology, we introduced large genomic DNA segments, up to 35 kbp, along with reporter genes into a newly designed vector with a potential cloning capacity of 46 kbp (Q4). Surprisingly, the insertion of diverse foreign DNAs alleviated the delayed plaque formation phenotype of Q4, and these large inserts remained stable through serial in vitro passages. With reporter-gene-expressing recombinant MCMVs, we successfully transduced not only mouse cell lines but also non-rodent mammalian cells, including those of human, monkey, bovine, and bat origin. Remarkably, even non-mammalian cell lines derived from chickens exhibited successful transduction.

A novel design method for wideband bandpass filters with fewer inductors

Inductors that are widely used in wideband filter designs could result in large filter size and high insertion loss. A novel design method using admittance matrix based on direct circuit implementation is introduced for wideband bandpass filters. Firstly, a frequency transformation is performed on cross-couplings to realize the conversion between inductor and capacitor, thereby reducing usage of inductors. Moreover, by virtue of an enhanced resonator (type I), an approach of adding extra transmission zero is proposed to improve the out-of-band rejection without any additional usage of inductors. Thirdly, based on the frequency transformation, another enhanced resonator (type II) is introduced to further improve the frequency characteristics without compromising usage of inductors. Finally, by cascading the enhanced resonators of type I and type II, a novel filter can be constructed. This novel filter is designed and fabricated using a glass-based integrated passive device (IPD) technology. The measurement results are highly consistent with the simulation results. It is shown that the proposed design method can reduce the filter size and thus improve the chip integration. It enriches the freedoms during the filter design.

Development of a Shuttle Vector That Transforms at High Frequency for the Emerging Human Fungal Pathogen: Candida auris.

Routine molecular manipulation of any organism is inefficient and difficult without the existence of a plasmid. Although transformation is possible in C. auris, no plasmids are available that can serve as cloning or shuttle vectors. C. auris centromeres have been well characterized but have not been explored further as molecular tools. We tested C. auris centromeric sequences to identify which, if any, could be used to create a plasmid that was stably maintained after transformation. We cloned all seven C. auris centromeric sequences and tested them for transformation frequency and stability. Transformation frequency varied significantly; however, one was found to transform at a very high frequency. A 1.7 Kb subclone of this sequence was used to construct a shuttle vector. The vector was stable with selection and maintained at ~1 copy per cell but could be easily lost when selection was removed, which suggested that the properties of the centromeric sequence were more Autonomously Replicating Sequence (ARS)-like than centromere-like when part of a plasmid. Rescue of this plasmid from transformed C. auris cells into E. coli revealed that it remained intact after the initial C. auris transformation, even when carrying large inserts. The plasmid was found to be able to transform all four clades of C. auris, with varying frequencies. This plasmid is an important new reagent in the C. auris molecular toolbox, which will enhance the investigation of this human fungal pathogen.

Genomic characterization revealing the high rate of tet(X4)-positive Escherichia coli in animals associated with successful genetic elements.

The rapid spread of plasmid-mediated tet(X4) conferring high tigecycline resistance poses a significant threat to public health. Escherichia coli as the most common pathogen which carries tet(X4) has been widely disseminated in China. Thus, comprehensive investigations are required to understand the mechanism of transmission of tet(X4)-positive E. coli. In this study, a total of 775 nonduplicate samples were collected in Guangdong, China from 2019 to 2020. We screened for tet(X4)-positive E. coli by PCR amplification and species identification. Furthermore, we analyzed the phylogenetics and genetic context of tet(X4)-positive E. coli through whole-genome sequencing and long-reads sequencing. Overall, 146 (18.84%) tet(X4)-positive E. coli were isolated, comprising 2 isolates from humans and 144 isolates from pigs. The majority of tet(X4)-positive E. coli exhibited resistance to multiple antibiotics but all of them were susceptible to amikacin and colistin. Phylogenetic analysis showed that ST877, ST871, and ST195 emerged as the predominant sequence types in tet(X4)-positive E. coli. Further analysis revealed various genetic environments associated with the horizontal transfer of tet(X4). Notably, a 100-kbp large fragment insertion was discovered downstream of tet(X4), containing a replicon and a 40-kbp gene cluster for the bacterial type IV secretion system. The high colonization rate of tet(X4)-positive E. coli in animals suggests that colonization as a key factor in its dissemination to humans. Diverse genetic context may contribute to the transfer of tet(X4). Our findings underline the urgent need for controlling the spread of plasmid-mediated tigecycline resistance.

Polarization rotator with large bandwidth and low insert loss based on lemon core photonic crystal fiber

Polarization rotator with large bandwidth and low insert loss based on lemon core photonic crystal fiber

Vaccinia Virus: Mechanisms Supporting Immune Evasion and Successful Long-Term Protective Immunity.

Vaccinia virus is the most successful vaccine in human history and functions as a protective vaccine against smallpox and monkeypox, highlighting the importance of ongoing research into vaccinia due to its genetic similarity to other emergent poxviruses. Moreover, vaccinia's ability to accommodate large genetic insertions makes it promising for vaccine development and potential therapeutic applications, such as oncolytic agents. Thus, understanding how superior immunity is generated by vaccinia is crucial for designing other effective and safe vaccine strategies. During vaccinia inoculation by scarification, the skin serves as a primary site for the virus-host interaction, with various cell types playing distinct roles. During this process, hematopoietic cells undergo abortive infections, while non-hematopoietic cells support the full viral life cycle. This differential permissiveness to viral replication influences subsequent innate and adaptive immune responses. Dendritic cells (DCs), key immune sentinels in peripheral tissues such as skin, are pivotal in generating T cell memory during vaccinia immunization. DCs residing in the skin capture viral antigens and migrate to the draining lymph nodes (dLN), where they undergo maturation and present processed antigens to T cells. Notably, CD8+ T cells are particularly significant in viral clearance and the establishment of long-term protective immunity. Here, we will discuss vaccinia virus, its continued relevance to public health, and viral strategies permissive to immune escape. We will also discuss key events and populations leading to long-term protective immunity and remaining key gaps.

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies.

Fosmids and cosmids are vectors frequently used in functional metagenomic studies. With a large insert capacity (around 30 kb) they can encode dozens of cloned genes or in some cases, entire biochemical pathways. Fosmids with cloned inserts can be transferred to heterologous hosts and propagated to enable screening for new enzymes and metabolites. After screening, fosmids from clones with an activity of interest must be de novo sequenced, a critical step toward the identification of the gene(s) of interest. In this work, we present a new approach for rapid and high-throughput fosmid sequencing directly from Escherichia coli colonies without liquid culturing or fosmid purification. Our sample preparation involves fosmid amplification with phi29 polymerase and then direct nanopore sequencing using the Oxford Nanopore Technologies system. We also present a bioinformatics pipeline termed "phiXXer" that facilitates both de novo read assembly and vector trimming to generate a linear sequence of the fosmid insert. Finally, we demonstrate the accurate sequencing of 96 fosmids in a single run and validate the method using two fosmid libraries that contain cloned large insert (~30-40 kb) genomic or metagenomic DNA.IMPORTANCELarge-insert clone (fosmids or cosmids) sequencing is challenging and arguably the most limiting step of functional metagenomic screening workflows. Our study establishes a new method for high-throughput nanopore sequencing of fosmid clones directly from lysed Escherichia coli cells. It also describes a companion bioinformatic pipeline that enables de novo assembly of fosmid DNA insert sequences. The devised method widens the potential of functional metagenomic screening by providing a simple, high-throughput approach to fosmid clone sequencing that dramatically speeds the pace of discovery.

Cymric, a Maternal and Zygotic HTK-16-Like SHARK Family Tyrosine Kinase Gene, Is Disrupted in Molgula occulta, a Tailless Ascidian.

AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, cymric (Uro-1), a HTK-16-like (HydraTyrosineKinase-16) gene, identified in a subtractive screen for maternal ascidian cDNAs in Molgula oculata, an ascidian species with a tadpole larva. The cymric gene encodes a 4-kb mRNA expressed in gonads, eggs, and embryos in the tailed M. oculata but is not detected in eggs or embryos of the closely related tailless species Molgula occulta. There is a large insertion in cymric in the M. occulta genome, as shown by transcriptome and genome analyses, resulting in it becoming a pseudogene. The cymric amino acid sequence encodes a nonreceptor tyrosine kinase with an N-terminal region containing two SH2 domains and five ankyrin repeats, similar to the HTK-16-like gene found in other ascidians. Thus, the ascidian cymric genes are members of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of nonreceptor tyrosine kinases, which are found throughout invertebrates and missing from vertebrates. We show that cymric is lacking the tyrosine kinase domain in the tailless M. occulta, although the truncated mRNA is still expressed in transcriptome data. This maternal and zygotic HTK-16-like tyrosine kinase is another described pseudogene from M. occulta and appears not to be necessary for adult development.