Transcriptome variation in human tissues revealed by long-read sequencing.

Regulation of transcript structure generates transcript diversity and plays an important role in human disease1-7. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8-16. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.

Somatic mutations and cell identity linked by Genotyping of Transcriptomes.

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.

Ancestral polymorphisms shape the adaptive radiation of Metrosideros across the Hawaiian Islands.

Some of the most spectacular adaptive radiations begin with founder populations on remote islands. How genetically limited founder populations give rise to the striking phenotypic and ecological diversity characteristic of adaptive radiations is a paradox of evolutionary biology. We conducted an evolutionary genomics analysis of genus Metrosideros, a landscape-dominant, incipient adaptive radiation of woody plants that spans a striking range of phenotypes and environments across the Hawaiian Islands. Using nanopore-sequencing, we created a chromosome-level genome assembly for Metrosideros polymorpha var. incana and analyzed whole-genome sequences of 131 individuals from 11 taxa sampled across the islands. Demographic modeling and population genomics analyses suggested that Hawaiian Metrosideros originated from a single colonization event and subsequently spread across the archipelago following the formation of new islands. The evolutionary history of Hawaiian Metrosideros shows evidence of extensive reticulation associated with significant sharing of ancestral variation between taxa and secondarily with admixture. Taking advantage of the highly contiguous genome assembly, we investigated the genomic architecture underlying the adaptive radiation and discovered that divergent selection drove the formation of differentiation outliers in paired taxa representing early stages of speciation/divergence. Analysis of the evolutionary origins of the outlier single nucleotide polymorphisms (SNPs) showed enrichment for ancestral variations under divergent selection. Our findings suggest that Hawaiian Metrosideros possesses an unexpectedly rich pool of ancestral genetic variation, and the reassortment of these variations has fueled the island adaptive radiation.

Assembly-free single-molecule sequencing recovers complete virus genomes from natural microbial communities.

Viruses are the most abundant biological entities on Earth and play key roles in host ecology, evolution, and horizontal gene transfer. Despite recent progress in viral metagenomics, the inherent genetic complexity of virus populations still poses technical difficulties for recovering complete virus genomes from natural assemblages. To address these challenges, we developed an assembly-free, single-molecule nanopore sequencing approach, enabling direct recovery of complete virus genome sequences from environmental samples. Our method yielded thousands of full-length, high-quality draft virus genome sequences that were not recovered using standard short-read assembly approaches. Additionally, our analyses discriminated between populations whose genomes had identical direct terminal repeats versus those with circularly permuted repeats at their termini, thus providing new insight into native virus reproduction and genome packaging. Novel DNA sequences were discovered, whose repeat structures, gene contents, and concatemer lengths suggest they are phage-inducible chromosomal islands, which are packaged as concatemers in phage particles, with lengths that match the size ranges of co-occurring phage genomes. Our new virus sequencing strategy can provide previously unavailable information about the genome structures, population biology, and ecology of naturally occurring viruses and viral parasites.

Controlled Synthesis and Surface Engineering of Janus Chitosan-Gold Nanoparticles for Photoacoustic Imaging-Guided Synergistic Gene/Photothermal Therapy.

The unsymmetrical morphology and unique properties of Janus nanoparticles (JNPs) provide superior performances for biomedical applications. In this work, a general and facile strategy is developed to construct a series of symmetrical and unsymmetrical chitosan/gold nanoparticles. Taking advantage of the active motion derived from Janus structure, selective surface functionalization of polysaccharide domain, and photothermal effect of gold nanorods, Janus chitosan/gold nanoparticles (J-Au-CS) are selected as a model system to construct Janus-structured chitosan/gold nanohybrids (J-ACP). Near-infrared (NIR)-responsive J-ACP composed of polycationic chitosan nanospheres and PEGylated gold nanorods hold great potential to realize photoacoustic (PA) imaging-guided complementary photothermal therapy (PTT)/gene therapy for breast cancer. The morphology effect of chitosan/gold nanostructures on enhanced PTT, cellular uptake, and gene transfection is investigated. The feasibility of PA imaging to track the accumulation of J-ACP and guide PTT is also explored. Notably, synergistic therapy is achieved based on PTT-enhanced gene therapy. In addition, the loading function of chitosan/gold nanoparticles for fluorescence imaging is demonstrated. The current work extends the application of JNPs for imaging-guided synergistic cancer therapy and provides flexible candidates with distinct structures for diverse biomedical applications.

Versatile Types of Organic/Inorganic Nanohybrids: From Strategic Design to Biomedical Applications.

Organic/inorganic nanohybrids have attracted widespread interests due to their favorable properties and promising applications in biomedical areas. Great efforts have been made to design and fabricate versatile nanohybrids. Among different organic components, diverse polymers offer unique avenues for multifunctional systems with collective properties. This review focuses on the design, properties, and biomedical applications of organic/inorganic nanohybrids fabricated from inorganic nanoparticles and polymers. We begin with a brief introduction to a variety of strategies for the fabrication of functional organic/inorganic nanohybrids. Then the properties and functions of nanohybrids are discussed, including properties from organic and inorganic parts, synergistic properties, morphology-dependent properties, and self-assembly of nanohybrids. After that, current situations of nanohybrids applied for imaging, therapy, and imaging-guided therapy are demonstrated. Finally, we discuss the prospect of organic/inorganic nanohybrids and highlight the challenges and opportunities for the future investigations.

Organic/inorganic nanohybrids as multifunctional gene delivery systems.

In this review, we summarize the rational design and versatile application of organic/inorganic hybrid gene carriers as multifunctional delivery systems. Organic/inorganic nanohybrids with both organic and inorganic components in one nanoparticle have attracted intense attention because of their favorable properties. Particularly, nanohybrids comprising cationic polymers and inorganic nanoparticles are considered to be promising candidates as multifunctional gene delivery systems. In this review, we begin with an introduction of gene delivery and gene carriers to demonstrate the incentive for fabricating nanohybrids as multifunctional carriers. Next, the construction strategies and morphology effects of organic/inorganic hybrid gene carriers are summarized and discussed. Both sections provide valuable information for the design and synthesis of hybrid gene carriers with superior properties. Finally, an overview is provided of the application of nanohybrids as multifunctional gene carriers. Diverse therapies and versatile imaging-guided therapies have been achieved via the rational design of nanohybrids. In addition to a simple combination of the functions of organic and inorganic components, the performances arising from the synergistic effects of both components are considered to be more intriguing. In summary, this review might offer guidance for the understanding of organic/inorganic nanohybrids as multifunctional gene delivery systems.

Dip-pen microarraying of molecular beacon probes on microgel thin-film substrates.

The integration of microarray-based nucleic acid detection technologies and microfluidics is attractive, because the combination of small sample volumes, relatively short diffusion distances, and solid-phase detection enhances the development of multiplexed assays with improved sensitivity and minimal sample size. However, traditional microarray spotting methods typically create probe spot sizes of ∼50-100 µm diameter, comparable to the dimensions of many microfluidic channels. In addition, detection of hybridization events typically requires a post-hybridization labeling step. We address both issues by exploring the use of dip-pen nanolithography (DPN) to pattern linear oligonucleotides and self-reporting molecular beacon (MB) probes on streptavidin-functionalized poly(ethylene glycol) microgel thin-film substrates. In contrast to many systems involving DPN deposition, the fluorescence of the labeled probes enables their amount and spatial distribution to be characterized by optical microscopy. Their deposition rate decreases with increasing DPN dwell time, consistent with a Langmuir adsorption model, but the linear relationship between spot diameter and time(1/2) indicates that spot size is diffusion controlled. We then use DPN to pattern MB probes for the mecA and spa genes in Staphylococcus aureus as a 2-column array with 1 µm spot sizes and 5 µm spot spacings, and we use this array to differentiate targets characteristic of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus. This duplexed self-reporting gel-tethered MB microarray not only shows high specificity but also a high signal-to-background ratio.

GoT-Splice protocol for multi-omics profiling of gene expression, cell-surface proteins, mutational status, and RNA splicing in human cells.

Studying RNA splicing factor mutations is challenging due to difficulties in distinguishing wild-type and mutant cells within complex human tissues and inaccuracies associated with reconstructing splicing signals from short-read sequencing data. Here, we present Genotyping of Transcriptomes (GoT)-Splice, a protocol that overcomes these limitations by combining GoT with enhanced long-read single-cell transcriptome and cell-surface proteomics profiling. We describe steps for long-read library preparation and analysis, followed by cDNA re-amplification, enrichment of mutation of interest, sample indexing, and GoT library preparation. For complete details on the use and execution of this protocol, please refer to Cortés-López et al.1.

High resolution long-read telomere sequencing reveals dynamic mechanisms in aging and cancer.

Telomeres are the protective nucleoprotein structures at the end of linear eukaryotic chromosomes. Telomeres' repetitive nature and length have traditionally challenged the precise assessment of the composition and length of individual human telomeres. Here, we present Telo-seq to resolve bulk, chromosome arm-specific and allele-specific human telomere lengths using Oxford Nanopore Technologies' native long-read sequencing. Telo-seq resolves telomere shortening in five population doubling increments and reveals intrasample, chromosome arm-specific, allele-specific telomere length heterogeneity. Telo-seq can reliably discriminate between telomerase- and ALT-positive cancer cell lines. Thus, Telo-seq is a tool to study telomere biology during development, aging, and cancer at unprecedented resolution.

Borg extrachromosomal elements of methane-oxidizing archaea have conserved and expressed genetic repertoires.

Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming "Candidatus Methanoperedens" archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding nanowire-like electron-transferring cytochromes and cell surface proteins are more highly expressed than those of host Methanoperedens, indicating that Borgs augment the Methanoperedens activity in situ. We reconstructed the first complete 4.00 Mbp genome for a Methanoperedens that is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enable Methanoperedens to distinguish their genomes from those of Borgs. Very high Borg to Methanoperedens ratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells.

Population Genomics of Nonrecessive Resistance to Bt Toxin Cry1Ac in Helicoverpa armigera From Northern China.

Transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) have provided control of some key pests since 1996. However, the evolution of resistance by pests reduces the benefits of Bt crops. Resistance to Bt crops that is not recessively inherited is especially challenging to manage. Here we analyzed nonrecessive resistance to Bt toxin Cry1Ac in eight field populations of Helicoverpa armigera sampled in 2018 from northern China, where this global pest has been exposed to Cry1Ac in Bt cotton since 1997. Bioassays revealed 7.5% of field-derived larvae were resistant to Cry1Ac of which 87% had at least one allele conferring nonrecessive resistance. To analyze this nonrecessive resistance, we developed and applied a variant of a genomic mapping approach called quantitative trait locus (QTL)-seq. This analysis identified a region on chromosome 10 associated with nonrecessive resistance to Cry1Ac in all 21 backcross families derived from field-collected moths. Individual sequencing revealed that all 21 field-collected resistant grandparents of the backcross families had a previously identified dominant point mutation in the tetraspanin gene HaTSPAN1 that occurs in the region of chromosome 10 identified by QTL-seq. QTL-seq also revealed a region on chromosome 26 associated with nonrecessive resistance in at most 14% of the backcross families. Overall, the results imply the point mutation in HaTSPAN1 is the primary genetic basis of nonrecessive resistance to Cry1Ac in field populations of H. armigera from northern China. Moreover, because nonrecessive resistance is predominant, tracking the frequency of this point mutation could facilitate resistance monitoring in the region.

Refuges of conventional host plants counter dominant resistance of cotton bollworm to transgenic Bt cotton.

Transgenic crops have revolutionized insect pest control, but evolution of resistance by pests threatens their continued success. The primary strategy for combating pest resistance to crops producing insecticidal proteins from Bacillus thuringiensis (Bt) uses refuges of non-Bt host plants to allow survival of susceptible insects. The prevailing paradigm is that refuges delay resistance that is rare and recessively inherited. However, we discovered refuges countered resistance to Bt cotton that was neither rare nor recessive. In a 15-year field study of the cotton bollworm, the frequency of a mutation conferring dominant resistance to Bt cotton surged 100-fold from 2006 to 2016 yet did not rise from 2016 to 2020. Computer simulations indicate the increased refuge percentage from 2016 to 2020 is sufficient to explain the observed halt in the evolution of resistance. The results also demonstrate the efficacy of a Bt crop can be sustained by non-Bt refuges of other crops.

Single-cell multi-omics defines the cell-type-specific impact of splicing aberrations in human hematopoietic clonal outgrowths.

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1mut erythroid progenitor cells. We uncovered distinct cryptic 3' splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 3' splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.

pH-Responsive hyaluronic acid-cloaked polycation/gold nanohybrids for tumor-targeted synergistic photothermal/gene therapy.

The combination of photothermal therapy (PTT) and gene therapy (GT) has attracted intense interest in cancer treatment. However, the lack of long circulation and active tumor targeting reduces the therapeutic efficacy of complementary PTT/GT. In this work, hyaluronic acid (HA)-cloaked gold nanorods-PGED (prepared by ring-opening of polyglycidyl methacrylate (PGMA) with ethylenediamine (ED))/pDNA (AP/pDNA-HA) complexes were prepared to achieve long circulation and tumor targeting for photoacoustic imaging (PAI)-guided synergistic PTT/GT. Gold nanorods endow the complexes with photothermal effect and PAI function. Benefiting from the HA cloak, the AP/pDNA-HA complexes exhibit excellent stability, biocompatibility, long circulation behavior and active targeting. In addition, the pH-responsive characteristic of the Schiff base bonds helps the AP/pDNA-HA complexes to effectively escape from the endosome/lysosome. The antioncogene p53 was employed to investigate the gene transfection efficiency of the delivery system both in vitro and in vivo. The superiority of synergistic PTT/GT is established in a mouse 4T1 breast tumor model. The current study provides a facile strategy for constructing multifunctional gene delivery systems with long circulation and tumor targeting features, which can achieve effective imaging-guided synergistic tumor treatment.

Identifying synergistic high-order 3D chromatin conformations from genome-scale nanopore concatemer sequencing.

High-order three-dimensional (3D) interactions between more than two genomic loci are common in human chromatin, but their role in gene regulation is unclear. Previous high-order 3D chromatin assays either measure distant interactions across the genome or proximal interactions at selected targets. To address this gap, we developed Pore-C, which combines chromatin conformation capture with nanopore sequencing of concatemers to profile proximal high-order chromatin contacts at the genome scale. We also developed the statistical method Chromunity to identify sets of genomic loci with frequencies of high-order contacts significantly higher than background ('synergies'). Applying these methods to human cell lines, we found that synergies were enriched in enhancers and promoters in active chromatin and in highly transcribed and lineage-defining genes. In prostate cancer cells, these included binding sites of androgen-driven transcription factors and the promoters of androgen-regulated genes. Concatemers of high-order contacts in highly expressed genes were demethylated relative to pairwise contacts at the same loci. Synergies in breast cancer cells were associated with tyfonas, a class of complex DNA amplicons. These results rigorously link genome-wide high-order 3D interactions to lineage-defining transcriptional programs and establish Pore-C and Chromunity as scalable approaches to assess high-order genome structure.

Biomineralized calcium carbonate nanohybrids for mild photothermal heating-enhanced gene therapy.

It is of great significance to develop multifunctional gene carriers to achieve treatments with enhanced therapeutic effects in an inflammation-free manner. In this work, assembled micelles of polysaccharide were utilized for the biomineralization of calcium carbonate to produce one-dimensional Alg-CaCO3 nanoparticles. In order to introduce both functions of mild hyperthermia and gene transfection, polydopamine (PDA) coating was applied to conjugate cationic polymers on the surface of nanoparticles. The resultant ACDP nanohybrids exhibited enhanced performance as gene carriers under near infrared (NIR) light irradiation at a low power density. Meanwhile, the pH-responsive degradation of gene carriers could further promote gene release for better effectiveness. The enhanced gene therapy induces tumor cell apoptosis, which could prevent inflammatory responses. The feasibility of mild hyperthermia-enhanced gene therapy for tumor treatment was investigated in vitro and in vivo. In addition, dual-modal ultrasound (US) and photoacoustic (PA) imaging was also realized to monitor and guide the treatment processes. The current work provides a new avenue for the construction of multifunctional platform to realize cancer therapy with improved therapeutic effectiveness in an inflammation-free manner.

Multiple origins of a single point mutation in the cotton bollworm tetraspanin gene confers dominant resistance to Bt cotton.

BACKGROUND: Transgenic crops producing insecticidal proteins derived from Bacillus thuringiensis (Bt) are used globally to kill key insect pests and provide numerous benefits, including improved pest management, increased profits, reduced insecticide use, and increased biological control. Unfortunately, such benefits are rapidly being lost by the evolution of Bt resistance by pests. RESULTS: The main strategy to delay resistance relies on the use of non-Bt refuge plants to produce sufficient susceptible insects that mate with rare resistant insects emerging from Bt crops, essentially diluting and/or removing resistance alleles from pest populations. A key assumption for the success of this refuge strategy is that inheritance of resistance is recessive. In China, dominant resistance to Cry1Ac Bt cotton by the cotton bollworm Helicoverpa armigera is increasing and is associated with a mutation in the tetraspanin HaTSPAN1 gene, conferring more than 125-fold resistance. Here, we used amplicon sequencing to test the hypotheses that the HaTSPAN1 mutation either arose from a single event and spread or that the mutation evolved independently several times throughout northern China. From three laboratory strains and 28 field populations sampled from northern China, we identified six resistant and 50 susceptible haplotypes. Phylogenetic analysis indicates that the HaTSPAN1 mutation arose from at least four independent origins and spread to their current distributions. CONCLUSION: The results provide valuable information about the evolutionary origins of dominant resistance to Cry1Ac Bt cotton in northern China and offer rationale for the rapid increase in field-evolved resistance in these areas, where the implementation of additional practical resistance management is needed. © 2020 Society of Chemical Industry.

Nanopore sequencing-based genome assembly and evolutionary genomics of circum-basmati rice.

BACKGROUND: The circum-basmati group of cultivated Asian rice (Oryza sativa) contains many iconic varieties and is widespread in the Indian subcontinent. Despite its economic and cultural importance, a high-quality reference genome is currently lacking, and the group's evolutionary history is not fully resolved. To address these gaps, we use long-read nanopore sequencing and assemble the genomes of two circum-basmati rice varieties. RESULTS: We generate two high-quality, chromosome-level reference genomes that represent the 12 chromosomes of Oryza. The assemblies show a contig N50 of 6.32 Mb and 10.53 Mb for Basmati 334 and Dom Sufid, respectively. Using our highly contiguous assemblies, we characterize structural variations segregating across circum-basmati genomes. We discover repeat expansions not observed in japonica-the rice group most closely related to circum-basmati-as well as the presence and absence variants of over 20 Mb, one of which is a circum-basmati-specific deletion of a gene regulating awn length. We further detect strong evidence of admixture between the circum-basmati and circum-aus groups. This gene flow has its greatest effect on chromosome 10, causing both structural variation and single-nucleotide polymorphism to deviate from genome-wide history. Lastly, population genomic analysis of 78 circum-basmati varieties shows three major geographically structured genetic groups: Bhutan/Nepal, India/Bangladesh/Myanmar, and Iran/Pakistan. CONCLUSION: The availability of high-quality reference genomes allows functional and evolutionary genomic analyses providing genome-wide evidence for gene flow between circum-aus and circum-basmati, describes the nature of circum-basmati structural variation, and reveals the presence/absence variation in this important and iconic rice variety group.

A flexible bowl-shaped magnetic assembly for multifunctional gene delivery systems.

Magnetic assemblies with special morphologies are promising for versatile biomedical applications due to their intriguing properties and performances. In this work, a polycation-functionalized bowl-shaped magnetic assembly (b-MNP-PGEA) was constructed for magnetic resonance imaging (MRI)-guided synergistic cancer therapy. Taking advantage of distinct properties of Fe3O4 nanoparticles, self-assembly concept, morphology control, and appropriate surface functionalization, the as-prepared magnetic assembly with special morphology was expected to work as a multifunctional carrier to realize the combination of magnetofection and photothermal therapy (PTT). The morphology effect of the magnetic assembly on cellular uptake and the subsequent gene transfection were investigated. The feasibility of the magnetic and photothermal carriers for MRI and complementary PTT/gene therapy was also studied. In addition, the excellent in vivo performance of the proposed bowl-shaped multifunctional carriers was demonstrated using a mouse breast cancer model. Interestingly, synergistic effects based on PTT-enhanced gene therapy were achieved. The facile assembly strategy for the development of special bowl-shaped magnetic carriers for synergistic PTT/gene therapy provides a new avenue for the versatile construction of efficient theranostic platforms.