Natural DNA Sequences Research Articles

Origins of fine structure in DNA melting curves.

With the help of the one-dimensional random Potts-like model, we study the origins of fine structures observed on differential melting profiles of double-stranded DNA. We theoretically assess the effects of sequence arrangement on DNA melting curves through the comparison of results for random, correlated, and block sequences. Our results re-confirm the smearing out of the fine structure with the increase in chain length for all types of sequence arrangements and suggest that the fine structure is a finite-size effect. We have found that the fine structures on melting curves of chains comprised of blocks with correlations in sequence are more persistent, probably because of increased sequence disorder the blocks introduce. Many natural DNAs show a well-expressed fine structure of melting profiles. Our results for block sequences may suggest the existence of such sequence motifs in natural DNA sequences.

DNA Quadruplex Structure with a Unique Cation Dependency.

DNA quadruplex structures provide an additional layer of regulatory control in genome maintenance and gene expression and are widely used in nanotechnology. We report the discovery of an unprecedented tetrastranded structure formed from a native G-rich DNA sequence originating from the telomeric region of Caenorhabditis elegans. The structure is defined by multiple properties that distinguish it from all other known DNA quadruplexes. Most notably, the formation of a stable so-called KNa-quadruplex (KNaQ) requires concurrent coordination of K+ and Na+ ions at two distinct binding sites. This structure provides novel insight into G-rich DNA folding under ionic conditions relevant to eukaryotic cell physiology and the structural evolution of telomeric DNA. It highlights the differences between the structural organization of human and nematode telomeric DNA, which should be considered when using C. elegans as a model in telomere biology, particularly in drug screening applications. Additionally, the absence/presence of KNaQ motifs in the host/parasite introduces an intriguing possibility of exploiting the KNaQ fold as a plausible antiparasitic drug target. The structure's unique shape and ion dependency and the possibility of controlling its folding by using low-molecular-weight ligands can be used for the design or discovery of novel recognition DNA elements and sensors.

NanoDeep: a deep learning framework for nanopore adaptive sampling on microbial sequencing.

Nanopore sequencers can enrich or deplete the targeted DNA molecules in a library by reversing the voltage across individual nanopores. However, it requires substantial computational resources to achieve rapid operations in parallel at read-time sequencing. We present a deep learning framework, NanoDeep, to overcome these limitations by incorporating convolutional neural network and squeeze and excitation. We first showed that the raw squiggle derived from native DNA sequences determines the origin of microbial and human genomes. Then, we demonstrated that NanoDeep successfully classified bacterial reads from the pooled library with human sequence and showed enrichment for bacterial sequence compared with routine nanopore sequencing setting. Further, we showed that NanoDeep improves the sequencing efficiency and preserves the fidelity of bacterial genomes in the mock sample. In addition, NanoDeep performs well in the enrichment of metagenome sequences of gut samples, showing its potential applications in the enrichment of unknown microbiota. Our toolkit is available at https://github.com/lysovosyl/NanoDeep.

Structural Optimization of Decoy Oligonucleotide-Based PROTAC That Degrades the Estrogen Receptor.

Proteolysis-targeting chimeras (PROTACs) have attracted attention as a chemical method of protein knockdown via the ubiquitin-proteasome system. Some oligonucleotide-based PROTACs have recently been developed for disease-related proteins that do not have optimal small-molecule ligands such as transcription factors. We have previously developed the PROTAC LCL-ER(dec), which uses a decoy oligonucleotide as a target ligand for estrogen receptor α (ERα) as a model transcription factor. However, LCL-ER(dec) has a low intracellular stability because it comprises natural double-stranded DNA sequences. In the present study, we developed PROTACs containing chemically modified decoys to address this issue. Specifically, we introduced phosphorothioate modifications and hairpin structures into LCL-ER(dec). Among the newly designed PROTACs, LCL-ER(dec)-H46, with a T4 loop structure at the end of the decoy, showed long-term ERα degradation activity while acquiring enzyme tolerance. These findings suggest that the introduction of hairpin structures is a useful modification of oligonucleotides in decoy oligonucleotide-based PROTACs.

Sequencing, Fast and Slow: Profiling Microbiomes in Human Samples with Nanopore Sequencing

Rapid and accurate pathogen identification is crucial in effectively combating infectious diseases. However, the current diagnostic tools for bacterial infections predominantly rely on century-old culture-based methods. Furthermore, recent research highlights the significance of host–microbe interactions within the host microbiota in influencing the outcome of infection episodes. As our understanding of science and medicine advances, there is a pressing need for innovative diagnostic methods that can identify pathogens and also rapidly and accurately profile the microbiome landscape in human samples. In clinical settings, such diagnostic tools will become a powerful predictive instrument in directing the diagnosis and prognosis of infectious diseases by providing comprehensive insights into the patient’s microbiota. Here, we explore the potential of long-read sequencing in profiling the microbiome landscape from various human samples in terms of speed and accuracy. Using nanopore sequencers, we generate native DNA sequences from saliva and stool samples rapidly, from which each long-read is basecalled in real-time to provide downstream analyses such as taxonomic classification and antimicrobial resistance through the built-in software (<12 h). Subsequently, we utilize the nanopore sequence data for in-depth analysis of each microbial species in terms of host–microbe interaction types and deep learning-based classification of unidentified reads. We find that the nanopore sequence data encompass complex information regarding the microbiome composition of the host and its microbial communities, and also shed light on the unexplored human mobilome including bacteriophages. In this study, we use two different systems of long-read sequencing to give insights into human microbiome samples in the ‘slow’ and ‘fast’ modes, which raises additional inquiries regarding the precision of this novel technology and the feasibility of extracting native DNA sequences from other human microbiomes.

NanoNIPT: Short-fragment nanopore sequencing of cell-free DNA for non-invasive prenatal testing of fetal aneuploidies and sex chromosome aberrations.

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DNA binding dynamics and target location mechanism of pioneer factor GAF.

The chromatin landscape regulates transcription by localizing and regulating the activity of sequence-specific DNA binding proteins, chromatin remodeling enzymes and transcription machinery. Pioneer transcription factors can target DNA motifs on closed chromatin and trigger local chromatin remodeling to increase chromatin accessibility. Despite this knowledge, how pioneer factors search for their cognate motif on chromatin remains poorly understood. Here, we use multicolor single-molecule fluorescence resonance energy transfer (smFRET) to investigate how a prototypical Drosophila pioneer factor GAF locates its cognate motif on chromatin. To investigate how GAF searches for its cognate motif on DNA and nucleosomes, we strategically placed fluorophores on the DNA binding domain of GAF (GAF-DBD) and DNA motifs, allowing distinction of motif-specific binding and nonspecific DNA binding by FRET. We observed GAF-DBD dwells on a DNA molecule containing one GAF binding motif for 3-4 seconds before dissociation. Interestingly, on a DNA sequence with two GAF binding motifs, GAF-DBD can slide between two motifs while staying bound to the DNA. This suggests GAF monomers use one-dimensional (1D) sliding to search for its motif on nucleosome-free DNA. We next investigated the scenario where one of the two motifs is protected by a nucleosome. If the motif is located at the edge of a 601 nucleosome (SHL7 location), GAF-DBD more frequently lands on the other motif on linker DNA before it slides to the nucleosomal motif. Nucleosomes effectively block 1D sliding when the motif is located more internally on the nucleosome, at SHL5 or SHL3. However, on a nucleosome reconstituted on a native Drosophila DNA sequence, GAF-DBD can directly target internal motifs by three-dimensional diffusion, instead of 1D sliding. Our findings reveal different search modes that can be used by pioneer factors to locate their cognate motifs on chromatin.

Abstract P008: Native Cell-Free DNA sequence analysis reliably infers epigenetically diverse subtypes of prostate cancer

Abstract Background: With the adoption of potent AR signaling inhibitors, there has been a prominent rise in the frequency of treatment-resistant tumor phenotypes. Highly aggressive transdifferentiated tumors may emerge as AR low or AR null prostate cancers (PCa) with or without neuroendocrine signatures. An accurate determination of these epigenetically diverse phenotypic subtypes has direct clinical implications, including therapy choice. Current approaches for disease subtype determination often require tissue biopsies, limiting due to the high risk of comorbidity. For a temporally evolving cancer, a minimally invasive molecular assay is urgently needed to determine disease subtypes accurately to guide clinical decisions. Methods & Results: Cell-free DNA assay emerging as a nextgen tool for cancer early detection and real-time tumor evolution monitoring tool. Cell-free DNA primarily consists of DNA protected by nucleosomes and DNA binding elements and is shed into the bloodstream by cells that undergo apoptosis. We hypothesized that a nucleosome protection map and transcriptional regulation could be reliably determined from circulating tumor DNA (ctDNA) analysis. This analysis can accurately classify advanced prostate cancer into epigenetically divergent subclasses such as adenocarcinoma (ARPC) and neuroendocrine (NEPC). For developing robust phenotype-specific signatures, we performed whole-genome sequencing on plasma cfDNA from 30 PDX lines to achieve moderately deep coverage (>10X) of “pure” human ctDNA sequence data. We applied an analysis framework “Griffin” to determine transcription factor binding sites and transcription start site nucleosome protection signatures, predictive of specific transcription factor activity. We also generated H3K27ac, H3K27me1, and H3K27me3 histone profiles from resected PDX tumors and observed that ctDNA fragmentation and coverage patterns are consistent with histone modifications. Guided by differential nucleosome profiles, we derived a novel ctDNA fragment size variability signature that accurately predicted expression deregulation for phenotype-defining genes. These signatures helped us build an unsupervised statistical model that accurately classified PCa subtypes. We applied our model to 2 clinical cohorts and attained 96% accuracy for classifying patients with well-defined subtypes of advanced PCa, using low coverage sequencing. Conclusion: We have developed a ctDNA analysis method that can accurately classify epigenetically diverse subtypes of advanced prostate cancer, spanning AR active and neuroendocrine subtypes. This scalable and cost-effective approach is also suitable for longitudinal monitoring and prediction of treatment-emergent prostate cancer resulting from therapeutic resistance. Citation Format: Navonil De Sarkar. Native Cell-Free DNA sequence analysis reliably infers epigenetically diverse subtypes of prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P008.

RF15 | PMON291 Crystal Structures of Androgen DNA Binding Domain Interacting with its Response Elements (C3(1) ARE and MMTV-GRE)

Abstract Prostate cancer (PCa) is the second leading cancer in the world in men. The major therapeutic target in advanced PCa is the ligand-binding pocket (LBP) of the androgen receptor (AR). The anti-androgens used to treat cancer are binding to this domain and compete with the natural agonist. Over time the cancer grows resistant towards this therapy. Nevertheless, the cancer still typically depends on the AR, indicating other sites of the AR may still serve as drug targets. The DNA binding domain (DBD) is a likely good therapeutic target as it is involved in essential processes such AR dimerization, translocation to the nucleus and binding to the androgen response elements (AREs). However the structure and the DNA binding interactions with AREs are not fully explored yet. Only one crystal structure (pdb: 1R4I) is available. In this structure, the DBD is bound to an artificial direct repeat DNA sequences (ADR3), resulting in an unexpected dimeric head-to-head arrangement. A better understanding on how the AR recognizes and binds to different native DNA sequences which are mainly organized as semi inverted repeats is crucial for rational drug design. Here, we performed protein crystallography of the DBD in complex with various natural ARE sequences, including the mouse mammary tumour virus (MMTV-GRE) motif. Surprisingly, this latter element is still able to be activated by a dimerization deficient AR. Our first crystal structures were hallmarked by a high degree of disorder and appeared to bind both in a head-to-head as a head-to-tail fashion. Rational engineering of the DNA duplexes used for crystallography yielded well diffracting crystals for two different complexes. These two structures were diffracted to a range of 2 to 2.7 Å. Both structures with natural sequences appeared to bind in the head-to-head arrangement including the MMTV-GRE which consist out of semi inverted repeat DNA, whereas the C3(1)ARE contains the canonical fully inverted repeats. The structure reveals a slightly bend arrangement from the previously reported structure. The displacement is most visible at the DNA recognition helix bound to the half site and the so-called lever arm, which positions this helix in this region with a RMSD difference of 1.3 A. This indicates that the structural conformation changes of the protein is driven towards improved DNA-base pair recognition. Indeed the protein is able to make an additional set of non-covalent interactions with the base pairs of MMTV-GRE, leading to higher affinity. Both these extra interactions and conformational change lead to a higher affinity binding of protein-DNA, which seems to overcome the barrier introduced by the dimerization breaking mutations. This interaction surface could be a druggable site for structure-based drug design in the future, targeting AR-DBD dimerization. Presentation: Sunday, June 12, 2022 12:30 p.m. - 12:35 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

PROTAC Degrader of Estrogen Receptor α Targeting DNA-Binding Domain in Breast Cancer.

PROteolysis-TArgeting Chimeras (PROTACs) are a powerful class of drugs that selectively degrade the proteins of interest (POIs) through cellular ubiquitination mechanisms. Estrogen receptor α (ERα) plays a vital role in the pathogenesis and treatment of breast cancer. In this work, the DNA-binding domain (DBD) of ERα was selected as the target to avoid drug resistance caused by the ligand-binding domain (LBD) of ERα. The estrogen response element (ERE), a natural DNA sequence binding with DBD of ERα, was chosen as a recognized unit of PROTAC. Therefore, we designed a nucleic acid-conjugated PROTAC, ERE-PROTAC, via a click reaction, in which the ERE sequence recruits ERα and the typical small molecule VH032 recruits the von Hippel-Lindau (VHL) E3 ligase. The proposed ERE-PROTAC showed to efficiently and reversibly degrade ERα in different breast cancer cells by targeting the DBD, indicating its potential to overcome the current resistance caused by LBD mutations.

In Vitro Mapping of Nucleosome Positions at Base-Pair Resolution Using Ortho-Phenanthroline.

The positions of nucleosomes along genomic DNA play a role in defining patterns of gene expression and chromatin organization. Determination of nucleosome positions in vivo and in vitro, as revealed by the locations of histones on DNA, has provided insight into mechanisms of nucleosome sliding, spacing, assembly, and disassembly. Here, we describe methods for the in vitro determination of histone-DNA contacts at base-pair (bp) resolution. The protocol involves the labeling of histones with ortho-phenanthroline (OP), site-specific cleavage of nucleosomal DNA, and processing and analysis of the resulting DNA fragments. This methodology provides an efficient and high-resolution means for studying kinetics and behavior of enzymes that alter nucleosome structure and/or positioning, and can be used to identify preferred distributions of nucleosomes on natural DNA sequences. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cysteine-specific chemical modification of folded histones with ortho-phenanthroline (OP) Basic Protocol 2: Nucleosome sliding assay adapted for OP mapping of histone-DNA contacts Basic Protocol 3: OP-mediated cleavage, processing, and analysis of DNA fragments using a sequencing gel Support Protocol 1: Preparation of dideoxy sequencing ladders Support Protocol 2: Preparation and running of a denaturing DNA sequencing gel.

Nanopore Sequencing for Detection and Characterization of Phosphorothioate Modifications in Native DNA Sequences.

Bacterial DNA is subject to various modifications involved in gene regulation and defense against bacteriophage attacks. Phosphorothioate (PT) modifications are protective modifications in which the non-bridging oxygen in the DNA phosphate backbone is replaced with a sulfur atom. Here, we expand third-generation sequencing techniques to allow for the sequence-specific mapping of DNA modifications by demonstrating the application of Oxford Nanopore Technologies (ONT) and the ELIGOS software package for site-specific detection and characterization of PT modifications. The ONT/ELIGOS platform accurately detected PT modifications in a plasmid carrying synthetic PT modifications. Subsequently, studies were extended to the genome-wide mapping of PT modifications in the Salmonella enterica genomes within the wild-type strain and strains lacking the PT regulatory gene dndB (ΔdndB) or the PT synthetic gene dndC (ΔdndC). PT site-specific signatures were observed in the established motifs of GAAC/GTTC. The PT site locations were in close agreement with PT sites previously identified using the Nick-seq technique. Compared to the wild-type strain, the number of PT modifications are 1.8-fold higher in ΔdndB and 25-fold lower in ΔdndC, again consistent with known regulation of the dnd operon. These results demonstrate the suitability of the ONT platform for accurate detection and identification of the unusual PT backbone modifications in native genome sequences.

A real-time one-time pad DNA-chaos image encryption algorithm based on multiple keys

Image encryption is an important necessity when sharing data over public networks, and many image encryption algorithms have been proposed for the task. However, most of these algorithms are complex and slow. We propose a real-time one-time pad DNA-chaos image encryption algorithm using multiple keys. The proposed encryption algorithm uses a greatly simplified mechanism and a large, unpredictable sequence to encrypt the image. Our algorithm has three main advantages. First, its reliance on the original image for four of the keys makes it sensitive to changes in the original image. Small changes in the image produce large changes in the keys, making the algorithm resistant to differential attacks. Second, we integrate the natural DNA sequence into the process of generating the key, and the algorithm uses the massive DNA sequences stored in the DNA gene bank to solve the problem that it is difficult to store one-time pad. The keys have the property of one-time pad, and the key space is considerablely large. Finally, our algorithm encrypts quickly enough to make it suitable for real-time applications. On the premise of ensuring that the results of this paper are not worse than those of the comparison literature in other performance indicators, in terms of encryption time, even if we use a computing environment that is worse than all the comparison literature computer configurations, the algorithm of this paper can still save at least 14%–86%. As the picture size becomes larger, the advantages of the algorithm in this paper are more obvious.

DNA coding and RDH scheme hybrid encryption algorithm using SVM

As the communication technology advanced rapidly in recent times, the need for confidential data communication also arose. Here, a computationally feasible encryption/decryption algorithm is proposed to secure data using DNA sequences. The principal objective of DNA algorithm is to reduce big image encryption time. In this algorithm, natural DNA sequences are used as main keys. The image in which secret data is hidden using reversible data hiding (RDH) technique is encrypted twice before transmission. RDH is an information security technology which is extremely helpful in telemedicine. Authentication is necessary for images captured by robots. This can be used for authentication of data. This technique also enables us to embed electronic patient records (EPR) data into medical image before transmission, which can be later recovered on transmission side. The images are divided block-wise before encryption in the proposed scheme. Machine learning helps us to design a support vector machine (SVM), based on which a classification scheme is obtained to group encrypted and original images separately and to recover original image from encrypted image.

Hybrid-architectured promoter design to deregulate expression in yeast.

Hybrid-architectured promoter design to deregulate expression in yeast under modulating power of carbon sources involves replacing native cis-acting DNA sequence(s) with de novo synthetic tools in coordination with master regulator transcription factor (TF) to alter crosstalk between signaling pathways, and consequently, transcriptionally rewire the expression. Hybrid-promoter architectures can be designed to mimic native promoter architectures in yeast's preferred carbon source utilization pathway. The method aims to generate engineered promoter variants (EPVs) that combine the advantages of being an exceptionally stronger EPV(s) than the naturally occurring promoters and permit "green-and-clean" production on a non-toxic carbon source. To implement the method, a predetermined essential part of the general transcription machinery is targeted. This targeting involves cis-acting DNA sequences to be replaced with synthetic cis-acting DNA sites in coordination with the targeted TF that must bind for transcription machinery activation. The method needs genomic and functional information that can lead to the discovery of the master TF(s) and synthetic cis-acting DNA elements, which enable the engineering of binding of master regulator TF(s). By introducing our recent work on the engineering of Pichia pastoris (syn. Komagataella phaffii) alcohol oxidase 1 (AOX1) hybrid-promoter architectures, we provide the method and protocol for the hybrid-architectured EPV design to deregulate expression in yeast. The method can be adapted to other promoters in different substrate utilization pathways in P. pastoris, as well as in other yeasts.

Advances in Fiber Optic DNA-Based Sensors: A Review

DNA is becoming increasingly important in the domain of optical fiber sensors, either as a tool for biosensing, or as a target to detect. In this review the main contributions of the last years are presented both in the domain of wavelength and intensity based configurations. This review comprises the use of natural single strand DNA (ssDNA) sequences as receptors for the detection of ssDNA sequences through hybridization, synthetic nucleic acids receptors for detection of complementary ssDNA sequences, and sensors based on natural and synthetic ssDNA receptors used for the detection of non-DNA targets. Parameters such as sensitivity, detection range and limit of detection are analyzed and discussed in detail to the purpose of comparing the different technologies and knowing the future lines to follow in the domain of fiber optic DNA-based sensors.

Addressing the role of centromere sites in activation of ParB proteins for partition complex assembly.

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.

Effects of Length and Loop Composition on Structural Diversity and Similarity of (G3TG3NmG3TG3) G-Quadruplexes.

A G-rich sequence containing three loops to connect four G-tracts with each ≥2 guanines can possibly form G-quadruplex structures. Given that all G-quadruplex structures comprise the stacking of G-quartets, the loop sequence plays a major role on their folding topology and thermal stability. Here circular dichroism, NMR, and PAGE are used to study the effect of loop length and base composition in the middle loop, and a single base difference in loop 1 and 3 on G-quadruplex formation of (G3HG3NmG3HG3) sequences with and without flanking nucleotides, where H is T, A, or C and N is T, A, C, or G. In addition, melting curve for G-quadruplex unfolding was used to provide relatively thermal stability of G-quadruplex structure after the addition of K+ overnight. We further studied the effects of K+ concentration on their stability and found structural changes in several sequences. Such (G3HG3NmG3HG3) configuration can be found in a number of native DNA sequences. The study of structural diversity and similarity from these sequences may allow us to establish the correlation between model sequences and native sequences. Moreover, several sequences upon interaction with a G-quadruplex ligand, BMVC, show similar spectral change, implying that structural similarity is crucial for drug development.

DNA punch cards for storing data on native DNA sequences via enzymatic nicking

Synthetic DNA-based data storage systems have received significant attention due to the promise of ultrahigh storage density and long-term stability. However, all known platforms suffer from high cost, read-write latency and error-rates that render them noncompetitive with modern storage devices. One means to avoid the above problems is using readily available native DNA. As the sequence content of native DNA is fixed, one can modify the topology instead to encode information. Here, we introduce DNA punch cards, a macromolecular storage mechanism in which data is written in the form of nicks at predetermined positions on the backbone of native double-stranded DNA. The platform accommodates parallel nicking on orthogonal DNA fragments and enzymatic toehold creation that enables single-bit random-access and in-memory computations. We use Pyrococcus furiosus Argonaute to punch files into the PCR products of Escherichia coli genomic DNA and accurately reconstruct the encoded data through high-throughput sequencing and read alignment.

Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing.

Background Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utility of DNA and RNA for prediction of antibiotic susceptibility. However, the effectiveness of bacterial direct RNA sequencing and base-calling has not previously been investigated. This study interrogated the genome and transcriptome of 4 extensively drug-resistant (XDR) K. pneumoniae clinical isolates; however, further antimicrobial susceptibility testing identified 3 isolates as pandrug-resistant (PDR).ResultsThe majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kb). DNA sequencing detected most resistance genes (≥70%) within 2 hours of sequencing. Neural network–based base-calling of direct RNA achieved up to 86% identity rate, although ≤23% of reads could be aligned. Direct RNA sequencing (with ∼6 times slower pore translocation) was able to identify (within 10 hours) ≥35% of resistance genes, including those associated with resistance to aminoglycosides, β-lactams, trimethoprim, and sulphonamide and also quinolones, rifampicin, fosfomycin, and phenicol in some isolates. Direct RNA sequencing also identified the presence of operons containing up to 3 resistance genes. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes.ConclusionOverall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.