Biology subjects

Ren, P.

Publications and source records attributed to Ren, P..

71 records · Page 4Linked to original sources

bioRxiv2021

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called Tiled-ClickSeq, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

molecular biology↗

bioRxiv2021

A trans-complementation system for SARS-CoV-2

The biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research and countermeasure development. Here we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing a deletion of ORF3 and envelope gene, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. The results suggest that the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.

microbiology↗

bioRxiv2021

Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

biophysics↗

bioRxiv2020

Design of intrinsically disordered proteins that undergo phase transitions with lower critical solution temperatures

Many naturally occurring elastomers are intrinsically disordered proteins (IDPs) built up of repeating units and they can demonstrate two types of thermoresponsive phase behavior. Systems characterized by lower critical solution temperatures (LCST) undergo phase separation above the LCST whereas systems characterized by upper critical solution temperatures (UCST) undergo phase separation below the UCST. There is congruence between thermoresponsive coil-globule transitions and phase behavior whereby the theta temperatures above or below which the IDPs transition from coils to globules serve as useful proxies for the LCST / UCST values. This implies that one can design sequences with desired values for the theta temperature with either increasing or decreasing radii of gyration above the theta temperature. Here, we show that the Monte Carlo simulations performed in the so-called intrinsic solvation (IS) limit version of the temperature-dependent the ABSINTH (self-Assembly of Biomolecules Studied by an Implicit, Novel, Tunable Hamiltonian) implicit solvation model, yields a useful heuristic for discriminating between sequences with known LCST versus UCST phase behavior. Accordingly, we use this heuristic in a supervised approach, integrate it with a genetic algorithm, combine this with IS limit simulations, and demonstrate that novel sequences can be designed with LCST phase behavior. These calculations are aided by direct estimates of temperature dependent free energies of solvation for model compounds that are derived using the polarizable AMOEBA (atomic multipole optimized energetics for biomolecular applications) forcefield. To demonstrate the validity of our designs, we calculate coil-globule transition profiles using the full ABSINTH model and combine these with Gaussian Cluster Theory calculations to establish the LCST phase behavior of designed IDPs.

biophysics↗

bioRxiv2020

TISCH: a comprehensive web resource enabling interactive single-cell transcriptome visualization of tumor microenvironment

Cancer immunotherapy targeting co-inhibitory pathways by checkpoint blockade shows remarkable efficacy in a variety of cancer types. However, only a minority of patients respond to treatment due to the stochastic heterogeneity of tumor microenvironment (TME). Recent advances in single-cell RNA-seq technologies enabled comprehensive characterization of the immune system heterogeneity in tumors, but also posed computational challenges on how to integrate and utilize the massive published datasets to inform immunotherapy. Here, we present Tumor Immune Single Cell Hub (TISCH, http://tisch.comp-genomics.org), a large-scale curated database that integrates single-cell transcriptomic profiles of nearly two million cells from 76 high-quality tumor datasets across 28 cancer types. All the data were uniformly processed with a standardized workflow, including quality control, batch effect removal, malignant cell classification, cell clustering, cell-type annotation, differential expression analysis, and functional enrichment analysis. TISCH provides interactive gene expression visualization across multiple datasets at the single-cell level or cluster level, allowing systematic comparison between different cell-types, patients, tissue origins, treatment and response groups, and even different cancer-types. In summary, TISCH provides a user-friendly interface for systematically visualizing, searching, and downloading gene expression atlas in the TME from multiple cancer types, enabling fast, flexible and comprehensive exploration of the TME.

bioinformatics↗

bioRxiv2020

Genetic signatures for lineage/sublineage classification of HPV16, 18, 52 and 58 variants

Increasing evidences indicate that high-risk HPV variants are heterogeneous in carcinogenicity and ethnic dispersion. In this work, we identified genetic signatures for convenient determination of lineage/sublineage of HPV16, 18, 52 and 58 variants. Using publicly available genomes, we found that E2 of HPV16, L2 of HPV18, L1 and LCR of HPV52, and L2, LCR and E1 of HPV58 contain the proper genetic signature for lineage/sublineage classification. Sets of hierarchical signature nucleotide positions (SNPs) were further confirmed for high accuracy (>98%) by classifying HPV genomes obtained from Chinese females, which included 117 HPV16 variants, 48 HPV18 variants 117 HPV52 variants and 89 HPV58 variants. The circulation of HPV variants posing higher cancer risk in Eastern China, such as HPV16 A4 and HPV58 A3, calls for continuous surveillance in this region. The marker genes and signature nucleotide positions may facilitate cost-effective diagnostic detections of HPV variants in clinical settings.

evolutionary biology↗

bioRxiv2020

Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2

The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Author summaryIn this study, we compared SARS-CoV-2 populations of 13 Chinese COVID-19 patients. Those viral populations contained a considerable proportion of viral sub-genomic messenger RNAs (sgmRNA), reflecting an active viral replication activity in the respiratory tract tissues. The comparison of 66 identified intra-host variants further showed a low viral genetic distance between intra-household patients and a narrow transmission bottleneck size. Despite the co-existence of genetically distinct viruses within the same host, most intra-host minor variants were not shared between transmission pairs, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Furthermore, the narrow bottleneck and active viral activity in the respiratory tract show that the passage of a small number of virions can cause infection. Our data have therefore delivered a key genomic resource for the SARS-CoV-2 transmission research and enhanced our understanding of the evolutionary dynamics of SARS-CoV-2.

genetics↗

bioRxiv2020

A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

A high-throughput platform would greatly facilitate COVID-19 serological testing and antiviral screening. Here we report a nanoluciferase SARS-CoV-2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. We demonstrate that the optimized reporter virus assay in Vero E6 cells can be used to measure neutralizing antibody activity in patient sera and produces results in concordance with a plaque reduction neutralization test (PRNT). Compared with the low-throughput PRNT (3 days), the SARS-CoV-2-Nluc assay has substantially shorter turnaround time (5 hours) with a high-throughput testing capacity. Thus, the assay can be readily deployed for large-scale vaccine evaluation and neutralizing antibody testing in humans. Additionally, we developed a high-throughput antiviral assay using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2). When tested against this reporter virus, remdesivir exhibited substantially more potent activity in A549-hACE2 cells compared to Vero E6 cells (EC50 0.115 vs 1.28 M), while this difference was not observed for chloroquine (EC50 1.32 vs 3.52 M), underscoring the importance of selecting appropriate cells for antiviral testing. Using the optimized SARS-CoV-2-Nluc assay, we evaluated a collection of approved and investigational antivirals and other anti-infective drugs. Nelfinavir, rupintrivir, and cobicistat were identified as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 M). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 M. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.

microbiology↗

bioRxiv2020

A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. Our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities.

microbiology↗

bioRxiv2020

Intra-host Variation and Evolutionary Dynamics of SARS-CoV-2 Population in COVID-19 Patients

As of middle May 2020, the causative agent of COVID-19, SARS-CoV-2, has infected over 4 million people with more than 300 thousand death as official reports1,2. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. Here, using deep sequencing data, we achieved and characterized consensus genomes and intra-host genomic variants from 32 serial samples collected from eight patients with COVID-19. The 32 consensus genomes revealed the coexistence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparison of allele frequencies of the iSNVs revealed genetic divergence between intra-host populations of the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events among intra-host transmissions. Nonetheless, we observed a maintained viral genetic diversity within GIT, showing an increased population with accumulated mutations developed in the tissue-specific environments. The iSNVs identified here not only show spatial divergence of intra-host viral populations, but also provide new insights into the complex virus-host interactions.

genomics↗

bioRxiv2020

Multiple approaches for massively parallel sequencing of HCoV-19 genomes directly from clinical samples

COVID-19 has caused a major epidemic worldwide, however, much is yet to be known about the epidemiology and evolution of the virus. One reason is that the challenges underneath sequencing HCoV-19 directly from clinical samples have not been completely tackled. Here we illustrate the application of amplicon and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of HCoV-19 from clinical samples covering a range of sample types and viral load. We also examine and compare the bias, sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. This is, to date, the first work systematically implements amplicon and capture approaches in sequencing HCoV-19, as well as the first comparative study across methods. Our work offers practical solutions for genome sequencing and analyses of HCoV-19 and other emerging viruses.

genomics↗

bioRxiv2020

Comparison of the two up-to-date sequencing technologies for genome assembly: HiFi reads of Pacbio Sequel II system and ultralong reads of Oxford Nanopore

The availability of reference genomes has revolutionized the study of biology. Multiple competing technologies have been developed to improve the quality and robustness of genome assemblies during the last decade. The two widely-used long read sequencing providers - Pacbio (PB) and Oxford Nanopore Technologies (ONT) - have recently updated their platforms: PB enable high throughput HiFi reads with base-level resolution with >99% and ONT generated reads as long as 2 Mb. We applied the two up-to-date platforms to one single rice individual, and then compared the two assemblies to investigate the advantages and limitations of each. The results showed that ONT ultralong reads delivered higher contiguity producing a total of 18 contigs of which 10 were assembled into a single chromosome compared to that of 394 contigs and three chromosome-level contigs for the PB assembly. The ONT ultralong reads also prevented assembly errors caused by long repetitive regions for which we observed a total 44 genes of false redundancies and 10 genes of false losses in the PB assembly leading to over/under-estimations of the gene families in those long repetitive regions. We also noted that the PB HiFi reads generated assemblies with considerably less errors at the level of single nucleotide and small InDels than that of the ONT assembly which generated an average 1.06 errors per Kb assembly and finally engendered 1,475 incorrect gene annotations via altered or truncated protein predictions.

genomics↗

bioRxiv2019

Enhancer-dependence of gene expression increases with developmental age

How overall principles of gene regulation (the \"logic\") may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic and topological profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1 and Myb occupancy. In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes employ distal enhancers for long-range regulation through enhancer-promoter looping, confirmed by Gata1 HiChIP. Genome editing demonstrated distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancer-dependence of transcription, we observed a progressive reliance on enhancer control with increasing age of ontogeny among diverse primary cells and tissues of mouse and human origin. Our findings highlight fundamental and conserved differences in regulatory logic at distinct developmental stages, characterized by simpler promoter-centric regulation in embryonic cells and combinatorial enhancer-driven control in adult cells.\n\nHighlightsO_LIRegulation of embryonic-specific erythroid genes is promoter-centric through Gata1\nC_LIO_LIAdult-specific control is combinatorial enhancer-driven and requires Myb\nC_LIO_LIAdult specific genes have increased enhancer-promoter chromatin interactions\nC_LIO_LIEnhancer-dependence increases progressively with increasing developmental age\nC_LI

molecular biology↗

bioRxiv2019

Comparative genomic analysis reveals a monophyletic cold adapted Arthrobacter cluster from polar and alpine regions

Decrease in the frequency of arginine and increase in lysine are the trends that have been identified in the genomes of cold adapted bacteria. However, some cold adapted taxa show only limited or no detectable changes in the frequencies of amino acid composition. Here, we examined Arthrobacter spp. genomes from a wide range of environments on whether the genomic adaptations can be conclusively identified across genomes of taxa from polar and alpine regions. Phylogenetic analysis with a concatenated alignment of 119 orthologous proteins revealed a monophyletic clustering of seven polar and alpine isolated strains. Significant changes in amino acid composition related to cold adaptation were exclusive to seven of the twenty-nine strains from polar and alpine regions. Analysis of significant indicator genes and cold shock genes also revealed that clear differences could only be detected in the same seven strains. These unique characteristics may result from a vast exchange of genome content at the node leading to the monophyletic cold adapted Arthrobacter cluster predicted by the birth-and-death model. We then experimentally validated that strains with significant changes in amino acid composition have a better capacity to grow at low temperature than the mesophilic strains.\n\nImportanceAcquisition of novel traits through horizontal gene transfer at the early divergence of the monophyletic cluster may accelerate their adaptation to low temperature. Our study reached a clear relationship between adaptation to cold and genomic features and would advanced in understanding the ambiguous results produced by the previous studies on genomic adaption to cold temperature.

microbiology↗

bioRxiv2019

Characterization of novel Chrysosporium morrisgordonii sp. nov., from bat white-nose syndrome (WNS) affected mines, northeastern United States

Psychrotolerant hyphomycetes including unrecognized taxon are commonly found in bat hibernation sites in Upstate New York. During a mycobiome survey, a new fungal species, Chrysosporium morrisgordonii sp. nov., was isolated from bat White-nose syndrome (WNS) afflicted Graphite mine in Warren County, New York. This taxon was distinguished by its ability to grow at low temperature spectra from 6{degrees}C to 25{degrees}C. Conidia were tuberculate and thick-walled, globose to subglobose, unicellular, 3.5-4.6 {micro}m x3.5-4.6 {micro}m, sessile or borne on narrow stalks. The stalks occurred at right angles to the hypha. Intercalary aleuroconidia were also observed; the hyphae were septate, hyaline, smooth-walled, 1-3 {micro}m wide, straight or branched. The ITS1-5.8S-ITS2 sequence of C. morrisgordonii differed from the closest match to C. speluncarum by 37 nucleotides (94% identity); the Neighbor-joining phylogenetic tree confirmed that C. morrisgordonii was placed in a well-supported monophyletic group with C. speluncarum. LSU and ITS phylogenetic trees further indicated that C. morrisgordonii, C. speluncarum, Renispora flavissima, Neogymnomyces virgineus and Gymnoascus demonbreunii formed a distinct group in the Onygenales.

microbiology↗

bioRxiv2018

Different subtypes of EGFR exon19 mutation can affect prognosis of patients with non-small cell lung adenocarcinoma

AimsIn this study, we determined whether different subtypes of epidermal growth factor receptor (EGFR) exon19 mutation are associated with the therapeutic effect of EGFR-tyrosine kinase inhibitors (TKIs) on advanced non-small cell lung adenocarcinoma.\n\nMethodsA total of 122 patients with stage III or IV non-small cell lung adenocarcinoma were retrospectively reviewed. Clinical characteristics of these patients, including progression-free survival (PFS) outcome for EGFR-TKI treatment, were analyzed.\n\nResultsAccording to the mutation pattern, we classified the in-frame deletions detected on EGFR Exon19 into three different types: codon deletion (CD), with a deletion of one or more original codons; codon substitution and skipping (CSS), with a deletion of one or two nucleotides but the residues could be translated into a new amino acid without changing following amino acid sequence; CD or CSS plus single nucleotide variant (SNV) (CD/CSS+SNV), exclude CD or CSS, theres another SNV nearby the deletion region. The clinical characteristics of three groups were analyzed and as a result, no significant difference was found. By comparing the average number of missing bases and amino acids of the three mutation subtypes, it could be discovered that the number of missing bases and amino acids of the three mutation subtypes is diverse, and group CSS> group CD> group CD/CSS+SNV. Finally, survival analysis was performed between three groups of patients. The median PFS of group CD, group CSS and group CD/CSS+SNV was 11 months, 9 months and 14 months respectively. There was a distinct difference in the PFS between group CSS and group CD/CSS+SNV (P=0.035<0.05), and the PFS of group CD/CSS+SNV was longer.\n\nConclusionsDifferent mutation subtypes of EGFR exon19 can predict the therapeutic effect of EGFR-TKIs on advanced non-small cell lung adenocarcinoma.

cancer biology↗

bioRxiv2017

Accurate Immune Repertoire Sequencing Reveals Malaria Infection Driven Antibody Lineage Diversification in Young Children

Accurately measuring antibody repertoire sequence composition in a small amount of blood is challenging yet important to the understanding of the repertoire response to infections and vaccinations. Here, we describe an accurate and high-coverage repertoire sequencing method, MIDCIRS, which uses as few as 1,000 naive B cells. Using it, we studied age-related antibody repertoire development and diversification before and during acute malaria in infants (< 12 months old) and toddlers (12 - 47 months old) with 4-8 ml of blood draws. Unexpectedly, we discovered high levels of somatic hypermutation (SHM) in infants as young as three months old. Antibody clonal lineage analysis revealed that both infants and toddlers increase SHM levels upon infection and memory B cells isolated from pre-malaria samples in malaria-experienced individuals continue to induce SHMs upon malaria rechallenge. These results highlight the vast potential of antibody repertoire diversification in infants and toddlers that has not been realized previously.

immunology↗