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Results for “Molecular Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2019

A generic approach for studying the kinetics of liquid-liquid phase separation under near-native conditions

Understanding the kinetics and underlying physicochemical forces of liquid-liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for the analysis of often severely aggregation-prone proteins. Frequently applied approaches, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, however, often lead to very different kinetic behaviors. Here we suggest that at extreme pH values even proteins such as the low-complexity domain (LCD) of hnRNPA2, TDP-43, and NUP-98 can be kept in solution, and then their LLPS can be induced by a jump to native pH, resulting in a system that can be easily controlled. This approach represents a generic method for studying LLPS under near native conditions, providing a platform for studying the phase-separation behavior of diverse proteins.

molecular biology↗

bioRxiv2022

Neryl acetate, the major component of Corsican Helichrysum Italicum essential oil, mediates its biological activities on skin barrier

Corsican Helichrysum Italicum essential oil is characterized by high concentrations of neryl acetate, and we previously demonstrated that Corsican Helichrysum Italicum essential oil increases the expression of genes that are part of the differentiation complex (involucrin, small proline rich proteins, late cornified envelope, S100 protein family). The biological activities of Helichrysum Italicum essential oil and neryl acetate were compared to identify how neryl acetate contributes to Helichrysum Italicum essential oil activity on human skin. Neryl acetate, as a part component of Helichrysum Italicum essential oil, was tested on skin explant models for 24 hours and 5 days in comparison with Helichrysum Italicum essential oil. We analyzed the biological regulations in the skin explant by transcriptomic analysis, skin barrier protein immunofluorescence, lipid staining and ceramide analysis by liquid chromatography-mass spectrometry. Transcriptomic analysis revealed that 41.5% of Helichrysum Italicum essential oil-modulated genes were also regulated by neryl acetate and a selected panel of genes were confirmed by quantitative reverse transcription PCR analysis. Those genes are involved in epidermal differentiation, skin barrier formation and ceramide synthesis. Involucrin, involved in formation of the cornified envelope, was upregulated at both gene and protein levels after 24 hours and 5 days respectively. After 5 days of treatment, total lipids and ceramides were also increased. Our results demonstrate that neryl acetate mediates a large part of Corsican Helichrysum Italicum essential oil activity on skin barrier formation.

molecular biology↗

bioRxiv2025

Structural basis and mechanism of action of NrdR, a bacterial master regulator of ribonucleotide reduction

Ribonucleotide reductases (RNRs) are the essential enzymes responsible for synthesizing dNTPs, the building blocks of DNA. In bacteria, the entire RNR network is controlled by the master regulator NrdR. As a regulator of an essential pathway with no eukaryotic equivalent, NrdR is a promising antimicrobial target. Recent studies have outlined a mechanism of action for NrdR, in which ATP and dATP induce changes in the protein quaternary structure, regulating RNR repression. However, due to a lack of functional studies linking the known structures to their biological roles, the role of NrdR is not yet fully understood. Here, we conducted a comprehensive study of NrdR in Escherichia coli and Pseudomonas aeruginosa. We delimited the NrdR regulon, combining transcriptomics and motif-based sequence analysis. We crystallized NrdR and obtained structural data on nucleotide binding and its relation to the stability of protein-protein interfaces involved in NrdR oligomerization. We examined the quaternary structures of NrdR using SEC-MALS and atomic force microscopy and correlated structure to function using point mutations, EMSAs, and in vitro transcription assays. Overall, our results demonstrate the mechanism used by NrdR to modulate its quaternary structure and activity and provide structural data that pave the way for targeted antimicrobial therapies.

molecular biology↗

bioRxiv2016

Transcriptomic Signatures for Ovulation in Vertebrates

Recently, we found anovulation in nuclear progestin receptor (Pgr) knockout (Pgr-KO) zebrafish, which offers a new model for examining Pgr regulated genes and pathways that are important for ovulation and fertility. In this study, we examined expression of all transcripts using RNA-Seq in pre-ovulatory follicular cells collected after the final oocyte maturation, but prior to ovulation, from wild-type (WT) or Pgr-KO fish. Differential expression analysis revealed 2,888 genes significantly differentially expressed between WT and Pgr-KO fish. Among those, 1,230 gene transcripts were significantly more expressed, while 1,658 genes were significantly less expressed in WT than those in Pgr-KO. We then retrieved and compared transcriptional data from online databases and further identified 661 conserved genes in fish, mice, and humans, that showed similar levels of high (283 genes) or low (387) expression in animals that were ovulating compared to those with no ovulation. For the first time, ovulatory genes and their involved biological processes and pathways were also visualized using Enrichment Map and Cytoscape. Intriguingly, enrichment analysis indicated the genes with higher expression were involved in multiple ovulatory pathways and processes such as inflammatory response, angiogenesis, cytokine production, cell migration, chemotaxis, MAPK, focal adhesion, and cytoskeleton reorganization. In contrast, the genes with lower expression were mainly involved DNA replication, DNA repair, DNA methylation, RNA processing, telomere maintenance, spindle assembling, nuclear acid transport, catabolic processes, nuclear and cell division. Our results indicate that a large set of genes (>3,000) are differentially regulated in the follicular cells in zebrafish prior to ovulation, terminating programs including growth and proliferation, and beginning processes including the inflammatory response and apoptosis. Further studies are required to establish relationships among these genes and an ovulatory circuit in zebrafish model.

Molecular Biology↗

bioRxiv2020

A streamlined, cost-effective, and specific method to deplete transcripts for RNA-seq

RNA-sequencing is a powerful and increasingly prevalent method to answer biological questions. Depletion of ribosomal RNA (rRNA), which accounts for 80% of total RNA, is an extremely important step to increase the power of RNA-seq. Selection for polyadenylated RNA is a commonly used approach that excludes rRNA, as well as, important non-polyadenylated RNAs, such as histones, circular RNAs, and many long noncoding RNAs. Commercial methods to deplete rRNA are cost-prohibitive and the gold standard method is no longer available as a standalone kit. Alternative non-commercial methods suffer from inconsistent depletion. Through careful characterization of all reaction parameters, we developed an optimized RNaseH-based depletion of human rRNA. Our method exhibited comparable or better rRNA depletion compared to commercial kits at a fraction of the cost and across a wide-range of input RNA amounts.

molecular biology↗

bioRxiv2020

Splicing modulators elicit global translational repression by condensate-prone proteins translated from introns

Chemical splicing modulators that bind to the spliceosome have provided an attractive venue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. Yet, the biological effect of proteins containing translated intron sequences remains unclear. Here we identified a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) using genome-wide ribosome profiling and bio-orthogonal non-canonical amino-acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through JNK phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.

molecular biology↗

bioRxiv2023

An optimized workflow for analyzing extracellular vesicles as biomarkers in liver diseases.

Background & AimsExtracellular vesicles (EVs) play an important role in intercellular communication, serving as vehicles for the exchange of biological materials and being involved in the regulation of physiological processes. EVs and their associated cargoes are considered a promising source of disease-associated biomarkers. The purpose of this study was to establish an easy-to-use, reproducible, and scalable workflow to efficiently analyze EVs in the context of liver disease. MethodsAn optimized workflow was established for the pre-analytical processing and isolation of EVs from plasma and serum. Nanoparticle Tracking Analysis (NTA) was used to characterize circulating EVs in the serum of patients with nonalcoholic fatty liver disease (NAFLD), autoimmune liver disease (AIH), and animal models with impaired liver function. EVs were separated from soluble proteins by an optimized, polyethylene glycol (PEG)-based enrichment protocol. Enriched EVs were either labeled and functionally characterized by monitoring cellular uptake or lysed for biomarker identification. ResultsCirculating EVs in the serum of patients with NAFLD or AIH and in different animal models have been characterized by NTA. Here we show that both the quantity and size of EVs in the serum of patients/animal models are significantly different from those of healthy individuals. We show that isolated EVs are functional, and their uptake by acceptor cells can be quantified after fluorescence labelling. Enriched EVs were directly used to analyze RNA biomarkers. Several microRNAs, including miR-15b, -16, -21, -122 and -223, were found to be significantly up-regulated in EVs isolated from the sera of patients with NAFLD and AIH. We show that EVs transport cytokines, and that IL-2, IL-6 and IL-8 were significantly up-regulated in EVs enriched from patients with cholangiocarcinoma (CCA) compared to healthy controls. ConclusionsThe workflow presented here represents an accessible and easy-to-use approach that enables the analysis and enrichment of EVs from complex biological fluids and their preparation for functional characterization or downstream analysis. In this study, the levels of several miRNAs were found to be significantly increased in EVs isolated from AIH and NAFLD patients compared with healthy controls. HighlightsO_LIEVs circulating in crude serum reflect the diseased stage of the donors. C_LIO_LIEnrichment of EVs with the approach presented here efficiently separates soluble proteins from EVs, providing optimal material for further characterization. C_LIO_LIExosomal markers are present in the EVs-enriched fraction. C_LIO_LIEnriched EVs are intact and are functionally taken up by acceptor cells. C_LIO_LIEnriched EVs are suitable, and have been used for, biomarkers identification both at RNA and protein level. C_LI

molecular biology↗

bioRxiv2023

Imaging membrane damage in ferroptosis and necrosis by wash-free fluorogenic chemical probes

Selectively labelling cells with damaged membranes is needed in contexts as simple as identifying dead cells in culture, or as complex as imaging membrane barrier functionality in vivo. The commonly used dyes are permanently coloured/fluorescent dyes that are simply excluded by intact membranes, but to achieve good image contrast therefore requires removing their extracellular signal by washing or background subtraction, which are not possible in vivo. Here, we develop fluorogenic probes which sensitively and selectively reveal damaged cells, without needing washing steps since their fluorescence turns on from near-zero background. From a set of novel fluorogenic probes impermeabilised by sulfonations along different vectors, we identify a specific disulfonated fluorogenic scaffold that enters cells only upon membrane damage, where it is enzymatically activated to mark them. The esterase probe iPS-FS2 is a reliable tool to reveal live cells that have been permeabilised by biological, biochemical, or physical membrane damage; and it can be used in multicolour microscopy. We confirm the modularity of this approach by also adapting it for redox-unmasked cell-excluded probes with improved hydrolytic stability. This scaffold-based design thus provides tools for wash-free in vivo imaging of membrane damage, which is relevant across many pathologies. The insightss gained from these probes should also be translatable to damage-targeted prodrugs, for selective therapy of membrane-compromised cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/543437v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1594016org.highwire.dtl.DTLVardef@7e7053org.highwire.dtl.DTLVardef@1008cb0org.highwire.dtl.DTLVardef@1148bd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2018

Distinct control of PERIOD2 degradation and circadian rhythms by the oncoprotein MDM2

The circadian clock relies on post-translational modifications to set the timing for degradation of core regulatory components and, thus, sets clock progression. Ubiquitin-modifying enzymes targeting clock components for degradation are known to mostly recognize phosphorylated substrates. A case in point is the circadian factor PERIOD 2 (PER2) whose phospho-specific turnover involves its recognition by {beta}-transducin repeat containing proteins ({beta}-TrCPs). Yet, the existence of this unique mode of regulation of PER2s stability falls short of explaining persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery.\n\nIn this study, we challenge the phosphorylation-centric view that PER2 degradation enhances circadian rhythm robustness by i) identifying the PER2:MDM2 endogenous complex, ii) establishing PER2 as a previously uncharacterized substrate for MDM2, iii) revealing an alternative phosphorylation-independent mechanism for PER2 ubiquitin-mediated degradation, iv) pinpointing residues for ubiquitin modification, and v) establishing the importance of MDM2-mediated PER2 turnover for defining the circadian period length. Our results not only expand MDM2s suite of specific substrates beyond the cell cycle to include circadian components but also uncover novel regulatory players that likely impact our view of how other mechanisms crosstalk and modulate the clock itself.

molecular biology↗

bioRxiv2025

Endogenous retrotransposon reverse transcriptase supports osteocyte function: implications for antiretroviral therapy-associated bone loss

Retrotansposable elements such as LINE-1 and HERV-K encode endogenous reverse transcriptases (RTs) with emerging roles in human biology. People living with HIV, particularly those receiving nucleoside reverse transcriptase inhibitor (NRTI)-based therapy, have increased risk of low bone mineral density (BMD), but the underlying mechanisms remain unclear. Here we show that endogenous RT activity, estimated by L1-Ta and HERV-K DNA content, is enriched in human bone and markedly reduced in osteoporosis, where it correlates with BMD and osteocyte gene expression. In vitro, nucleoside reverse transcriptase inhibitors (NRTIs), widely used as antiretroviral drugs, impair osteocyte endocrine function, disrupting vitamin D3-induced FGF23 production and parathyroid hormone- mediated regulation of SOST. These findings identify endogenous RT activity as a regulator of osteocyte function and bone homeostasis, and suggest that its pharmacological inhibition may contribute to antiretroviral therapy-associated bone loss.

molecular biology↗

bioRxiv2025

Heme Detoxification in the Malaria Parasite Plasmodium falciparum: A Time-Dependent Basal-Level Analysis

Malaria is a deadly disease for which therapeutic options are threatened by the rise of antimalarial resistance. Inhibiting the formation of hemozoin (the product of heme detoxification) in the digestive vacuole (DV) is the mechanism of action of numerous antimalarial drugs, including those in development as new therapies. This drug target remains attractive as hemozoin is an abiotic and non-mutable molecule, unique to the parasite. The underlying parasite biology of the heme detoxification pathway is complex and requires a deeper understanding. This study focuses on the DV of Plasmodium falciparum, utilizing confocal microscopy, immunoblotting and cellular fractionation techniques to study its native state over time. Using parameters such as the uptake into and growth of the DV, relative abundance of plasmepsins (PMs) I and IV and basal levels of hemoglobin, heme and hemozoin, it was found that DV physiology in chloroquine (CQ)-sensitive NF54 parasites follows three distinct developmental phases: the lag-type growth (20 to 28 h), rapid growth phase (28 to 40 h) and the plateau (40 to 48 h). These phases hold specific characteristics with respect to the investigated parameters. In addition, key differences between CQ-sensitive NF54 and CQ-resistant Dd2 parasites were observed.

molecular biology↗

bioRxiv2019

Comparison of visceral adipose tissue DNA methylation and gene expression profiles in female adolescents with obesity

BackgroundEpigenetic changes in visceral adipose tissue (VAT) with obesity and their effects on gene expression are poorly understood, especially during emergent obesity in youth. The current study tested the hypothesis that methylation and gene expression profiles of key growth factor and inflammatory pathways such as PI3K/AKT signaling are altered in VAT from obese compared to non-obese youth.\n\nMethodsVAT samples from adolescent females grouped as Lean (L; n=15; age=15{+/-}3 yrs, BMI=21.9{+/-}3.0 kg/m2) or Obese (Ob; n=15, age=16{+/-}2 yrs, BMI=45.8{+/-}9.8 kg/m2) were collected. Global methylation (n=20) and gene expression (N=30) patterns were profiled via microarray and interrogated for differences between groups by ANCOVA (p<0.05), followed by biological pathway analysis.\n\nResultsOverlapping differences in methylation and gene expression in 317 genes were found in VAT from obese compared to lean groups. PI3K/AKT Signaling (p=1.83x10-6; 10/121 molecules in dataset/pathway) was significantly overrepresented in Ob VAT according to pathway analysis. mRNA upregulations in the PI3K/AKT Signaling Pathway genes TFAM (p=0.03; Fold change=1.8) and PPP2R5C (p=0.03, FC=2.6) were confirmed via qRT-PCR.\n\nConclusionOur analyses show obesity-related differences in DNA methylation and gene expression in visceral adipose tissue of adolescent females. Specifically, we identified methylation site/gene expression pairs differentially regulated and mapped these differences to PI3K/AKT signaling, suggesting that PI3K/AKT signaling pathway dysfunction in obesity may be driven in part by obesity-related changes in DNA methylation.

molecular biology↗

bioRxiv2021

A Scalable Screening of E. coli Strains for Recombinant Protein Expression

Structural biology projects are highly dependent on the large-scale expression of soluble protein and, for this purpose, heterologous expression using bacteria or yeast as host systems is usually employed. In this scenario, some of the parameters to be optimized include (i) those related to the protein construct, such as the use of a fusion protein, the choice of an N-terminus fusion/tag or a C-terminus fusion/tag; (ii) those related to the expression stage, such as the concentration and selection of inducer agent and temperature expression and (iii) the choice of the host system, which includes the selection of a prokaryotic or eukaryotic cell and the adoption of a strain. The optimization of some of the parameters related to protein expression, stage (ii), is straightforward. On the other hand, the determination of the most suitable parameters related to protein construction requires a new cycle of gene cloning, while the optimization of the host cell is less straightforward. Here, we evaluated a scalable approach for the screening of host cells for protein expression in a structural biology pipeline. We evaluated four Escherichia coli strains looking for the best yield of soluble heterologous protein expression using the same strategy for protein construction and gene cloning and comparing it to our standard strain, Rosetta 2 (DE3). Using a liquid handling device (robot), E. coli pT-GroE, Lemo21(DE3), Arctic Express (DE3), and Rosetta Gami 2 (DE3) strains were screened for the maximal yield of soluble heterologous protein recovery. For the genes used in this experiment, the Arctic Express (DE3) strain resulted in better yields of soluble heterologous proteins. We propose that screening of host cell/strain is feasible, even for smaller laboratories and the experiment as proposed can easily be scalable to a high-throughput approach.

molecular biology↗

bioRxiv2022

A mass spectrometry-based atlas of extracellular matrix proteins across 25 mouse organs

The extracellular matrix is a critical non-cellular component of multi-cellular organisms containing a variety of proteins, glycoproteins, and proteoglycans which has been implicated in a wide variety of essential biological processes, including development, wound healing, and aging. Due to low solubility, many ECM proteins have been underrepresented in previous proteomics datasets. Using an optimized 3-step decellularization and ECM extraction method involving chaotrope extraction and digestion via hydroxylamine hydrochloride, we have generated coverage of the matrisome across 25 organs. We observe that the top 100 most abundant proteins from the ECM fractions of all tissues are generally present in all tissues, indicating that tissue matrices are principally composed of a shared set of ECM proteins. However, these proteins vary up to 4,000-fold between tissues, resulting in highly unique matrix profiles even with the same primary set of proteins. A data reduction approach was used to reveal related networks of expressed ECM proteins across varying tissues, including basement membrane and collagen subtypes.

molecular biology↗

bioRxiv2023

Development of a multifunctional toolkit of intrabody-based biosensors recognizing the V5 peptide tag: highlighting applications with G protein-coupled receptors

ABSTRACT/SUMMARYProtein-protein interactions (PPIs) form the underpinnings of any cellular signaling network. PPIs are highly dynamic processes and often, cell-based assays can be essential for their study as they closely mimic the biological intricacies of cellular environments. Since no sole platform can perform all needed experiments to gain a thoroughly comprehensive understanding into these processes, developing a versatile toolkit is much needed to address this longstanding gap. The use of small peptide tags, such as the V5-tag, has been extensively used in biological and biomedical research, including labeling the C-termini of one of the largest human genome-wide open-reading frame collections. However, these small peptide tags have been primarily used in vitro and lack the in vivo traceability and functionality of larger specialized tags. In this study, we combined structural studies and computer-aided maturation to generate an intracellular nanobody, interacting with the V5-tag. Suitable for assays commonly used to study protein-protein interactions, our nanobody has been applied herein to interrogate G protein-coupled receptor signalling. This novel serviceable intrabody is the cornerstone of a multipurpose intracellular nanobody-based biosensors toolkit, named iBodyV5, which will be available for the scientific community at large.

molecular biology↗

bioRxiv2020

Regulation of the one carbon folate cycle as a shared metabolic signature of longevity

The metabolome represents a complex network of biological events that reflects the physiologic state of the organism in heath and disease. Additionally, specific metabolites and metabolic signaling pathways have been shown to modulate animal ageing, but whether there are convergent mechanisms uniting these processes remains elusive. Here, we used high resolution mass spectrometry to obtain the metabolomic profiles of canonical longevity pathways in C. elegans and identify metabolites regulating life span. By leveraging the metabolomic profiles across pathways, we found that one carbon metabolism and the folate cycle were pervasively regulated in common. We observed similar changes in long lived mouse models of reduced insulin/IGF signaling. Genetic manipulation of pathway enzymes and supplementation with one carbon metabolites reveal that regulation of the folate cycle represents a shared causal mechanism of longevity and proteoprotection.

molecular biology↗

bioRxiv2023

Jointly modeling deep mutational scans identifies shifted mutational effects among SARS-CoV-2 spike homologs

Deep mutational scanning (DMS) is a high-throughput experimental technique that measures the effects of thousands of mutations to a protein. These experiments can be performed on multiple homologs of a protein or on the same protein selected under multiple conditions. It is often of biological interest to identify mutations with shifted effects across homologs or conditions. However, it is challenging to determine if observed shifts arise from biological signal or experimental noise. Here, we describe a method for jointly inferring mutational effects across multiple DMS experiments while also identifying mutations that have shifted in their effects among experiments. A key aspect of our method is to regularize the inferred shifts, so that they are nonzero only when strongly supported by the data. We apply this method to DMS experiments that measure how mutations to spike proteins from SARS-CoV-2 variants (Delta, Omicron BA.1, and Omicron BA.2) affect cell entry. Most mutational effects are conserved between these spike homologs, but a fraction have markedly shifted. We experimentally validate a subset of the mutations inferred to have shifted effects, and confirm differences of >1,000-fold in the impact of the same mutation on spike-mediated viral infection across spikes from different SARS-CoV-2 variants. Overall, our work establishes a general approach for comparing sets of DMS experiments to identify biologically important shifts in mutational effects. Significance StatementAmino-acid mutations to a protein have effects that can shift as the protein evolves or is put under new selective pressure. The effects of amino-acid mutations to a specific protein under a defined selective pressure can be measured by deep mutational scanning experiments. Here, we devise an approach to quantify shifts in mutational effects between experiments performed on different homologs (i.e. variants) of the same protein, or on the same protein selected under different conditions. We use this approach to compare experiments performed on three homologs of SARS-CoV-2 spike, identifying mutations that have shifted in their effect on spike-mediated viral infection by >1,000 fold across SARS-CoV-2 variants.

molecular biology↗

bioRxiv2024

Comparison of direct cDNA and PCR-cDNA Nanopore sequencing of Escherichia coli isolates

2.Whole-transcriptome (long-read) RNA sequencing (Oxford Nanopore Technologies, ONT) holds promise for agnostic analysis of differential gene expression (DGE) in pathogenic bacteria, including for antimicrobial resistance genes (ARGs). However, direct cDNA ONT sequencing requires large concentrations of polyadenylated mRNA, and amplification protocols may introduce technical bias. Here we evaluated the impact of direct cDNA and cDNA PCR-based ONT sequencing on transcriptomic analysis of clinical Escherichia coli. Four E. coli bloodstream infection-associated isolates (n=2 biological replicates/isolate) were sequenced using the ONT Direct cDNA Sequencing SQK-DCS109 and PCR-cDNA Barcoding SQK-PCB111.24 kits. Biological and technical replicates were distributed over 8 flow cells using 16 barcodes to minimise batch/barcoding bias. Reads were mapped to a transcript reference and transcript abundance quantified after in silico depletion of low abundance and rRNA genes. We found there were strong correlations between read counts using both kits and when restricting the analysis to include only ARGs. We highlighted correlations were weaker for genes with a higher GC content. Read lengths were longer for the direct cDNA kit compared to the PCR-cDNA kit whereas total yield was higher for the PCR-cDNA kit. In this small but methodologically rigorous evaluation of biological and technical replicates of isolates sequenced with the direct cDNA and PCR-cDNA ONT sequencing kits, we demonstrated that PCR-based amplification substantially improves yield with largely unbiased assessment of core gene and ARG expression. However, users of PCR-based kits should be aware of a small risk of technical bias which appears greater for genes with an unusually high (>52%)/low (<44%) GC-content. 3. Impact statementRNA sequencing allows quantification of RNA within a biological sample providing information on the expression of genes at a particular time. This helps understand the expression of antimicrobial resistance genes (ARGs). In RNA-Seq experimental workflows extra steps of reverse transcription may be needed to generate more stable cDNA to allow for amplification by PCR if starting RNA input was low. Two current methods of long-read RNA sequencing include direct cDNA and PCR-cDNA based sequencing (Oxford Nanopore Technologies, ONT). However, few studies have compared these two methods of RNA-sequencing using clinical bacterial isolates. We therefore undertook a study to compare both kits using a methodological balanced design of biological and technical replicates of E. coli. Our study showed that direct cDNA and PCR-cDNA sequencing is highly reproducible between biological and technical E. coli replicates with very small differences in gene expression signatures generated between kits. The PCR-cDNA kit generates increased sequencing yield but a smaller proportion of mappable reads, the generation of shorter reads of lower quality and some PCR-associated bias. PCR-based amplification greatly increased sequencing yield of core genes and ARGs, however there may be a small risk of PCR-bias in genes that have a higher GC content. 4. Data summaryThe transcript reads of the four sequenced Escherichia coli strains have been deposited in the Figshare, DOI: 10.6084/m9.figshare.25044051. The authors confirm all supporting data (available in Figshare), code (available at: https://github.com/samlipworth/rna_methods) and protocols have been provided within the article or through supplementary data files.

molecular biology↗