Single DNA Molecules Research Articles

Deciphering the Structural Variants in B Cell Acute Lymphoblastic Leukemia By Optical Genome Mapping

Introduction Structural variants (SVs) of the genome play a key role in the leukemogenesis and development of B cell lymphoblastic leukemia (B-ALL), including gene deletion, amplification, translocation, and other forms. Methods for analyzing SVs and CNVs include karyotype analysis, fluorescence in situ hybridization, gene chips, and high-throughput gene sequencing technologies. Optical genome mapping (OGM) is a novel ultra-long single-molecule DNA detection based high throughput SVs analysis technology, which has the characteristics of whole genome coverage, high detection incidence and sensitivity. There were few publications presenting technical concordance of SVs detection beween karyotyping and OGM, and there are no systematic studies comparing the concordance of OGM and RNA-seq in detecting gene fusion. This study aimed to compare the concordance of OGM and RNA-seq in the detection of fusion genes in B-ALL cases, and the additional findings using OGM technology. Methods Bone marrow aspirates of 70 patients with newly diagnosed or relapsed/refractory B-ALL were recruited into the study. All samples were conducted with both OGM and transcriptome sequencing (RNA-seq). Among these cases, 42 males and 28 females, 30 pediatric (< 18 years) and 40 adults (≥ 18 years) cases, ages ranged 4-60 years (median 16 years). Results Totally 50 gene fusions were reported in 48 cases, with 2 cases each carrying two fusion genes ( BCR::ABL1/ PAX5::ELK3 and P2RY8::CRLF2/ PAX5::GSE1, respectively) (Figure 1A). OGM and RNA-seq have complete concordance in reporting gene fusions, and OGM provides further insight into the genomic events that lead to gene fusion. A subtype of B-ALL characterized by UBTF microdeletion leading to UBTF::ATXN7L3 fusion and PAN3 downstream deletion-induced CDX2 aberrant expression has been reported recently, and has been accepted by the International Consensus Classification as a novel molecular subtype of UBTF/CDX2 of B-ALL. We performed the OGM investigation on 6 UBTF/CDX2 cases which were identified by RNA-seq. OGM identified both microdeletion events in all 6 cases, and further reported 1q amplification in 3 of them (Figure 1B, C). B-lymphocyte differentiation-associated transcription factor gene defects are common and important genetic abnormalities in B-ALL, often manifested as complete or partial deletion of genes. We focused on a panel including 30 genes in the OGM data and determined IKZF1 deletion in 35 patients, CDKN2A/B deletion in 32 patients, and PAX5 deletion in 25 patients (Figure 1 A). Follow-up data showed that patients with CDKN2A/B deletion had a worse prognosis than those without CDKN2A/B deletion ( P = 0.025). Conclusion This is the first B-ALL cohort to systematically compare OGM and RNA-seq in gene fusion detection, and the result showed complete consistency. RNA-seq has significant advantages in analyzing gene fusion transcripts, while OGM has technical advantages in analyzing SVs at the genome level. Combining the two technologies can accurately analyze gene fusion and other SVs in B-ALL, significantly improve the detection rate of SVs with important pathological/diagnostic classification significance, and may bring new discoveries on SVs and pathogenic mechanisms.

Fluorescence Microscopy of Nanochannel-Confined DNA.

Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level, and the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments, and analyze the data.

Genome-wide single-molecule analysis of long-read DNA methylation reveals heterogeneous patterns at heterochromatin that reflect nucleosome organisation.

High-throughput sequencing technology is central to our current understanding of the human methylome. The vast majority of studies use chemical conversion to analyse bulk-level patterns of DNA methylation across the genome from a population of cells. While this technology has been used to probe single-molecule methylation patterns, such analyses are limited to short reads of a few hundred basepairs. DNA methylation can also be directly detected using Nanopore sequencing which can generate reads measuring megabases in length. However, thus far these analyses have largely focused on bulk-level assessment of DNA methylation. Here, we analyse DNA methylation in single Nanopore reads from human lymphoblastoid cells, to show that bulk-level metrics underestimate large-scale heterogeneity in the methylome. We use the correlation in methylation state between neighbouring sites to quantify single-molecule heterogeneity and find that heterogeneity varies significantly across the human genome, with some regions having heterogeneous methylation patterns at the single-molecule level and others possessing more homogeneous methylation patterns. By comparing the genomic distribution of the correlation to epigenomic annotations, we find that the greatest heterogeneity in single-molecule patterns is observed within heterochromatic partially methylated domains (PMDs). In contrast, reads originating from euchromatic regions and gene bodies have more ordered DNA methylation patterns. By analysing the patterns of single molecules in more detail, we show the existence of a nucleosome-scale periodicity in DNA methylation that accounts for some of the heterogeneity we uncover in long single-molecule DNA methylation patterns. We find that this periodic structure is partially masked in bulk data and correlates with DNA accessibility as measured by nanoNOMe-seq, suggesting that it could be generated by nucleosomes. Our findings demonstrate the power of single-molecule analysis of long-read data to understand the structure of the human methylome.

A framework to validate fluorescently labeled DNA-binding proteins for single-molecule experiments

Due to the enhanced labeling capability of maleimide-based fluorescent probes, lysine-cysteine-lysine (KCK) tags are frequently added to proteins for visualization. In this study, we employed an invitro single-molecule DNA flow-stretching assay as a sensitive way to assess the impact of the KCK tag on the property of DNA-binding proteins. Using Bacillus subtilis ParB as an example, we show that, although no noticeable changes were detected by invivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays, the KCK tag substantially altered ParB's DNA compaction rates and its response to nucleotide binding and to the presence of the specific sequence (parS) on the DNA. While it is typically assumed that short peptide tags minimally perturb protein function, our results urge researchers to carefully validate the use of tags for protein labeling. Our comprehensive analysis can be expanded and used as a guide to assess the impacts of other tags on DNA-binding proteins in single-molecule assays.

Mind your tag in single-molecule measurements

In this issue of Cell Reports Methods, Molina and colleagues use invitro single-molecule DNA flow-stretching to demonstrate the severe effects of appending a short lysine-cysteine-lysine (KCK) tag on the Bacillus subtilis ParB protein. This assay could be further utilized to evaluate the impact of other tags on DNA-binding proteins.

Secondary Metabolite Analysis of Phomopsis sp. LGT-5 Based on the Dual Guidance of Whole-Genome Sequencing and HPLC-Q-ToF-MS/MS.

Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities. The Phomopsis sp. LGT-5 was obtained through tissue block and repeatedly crossed methods from Tripterygium wilfordii Hook. F. The antibacterial experiments of LGT-5 showed that it has high inhibitory activity against Staphylococcus aureus and Pseudomonas aeruginosa, and moderate inhibitory activity against Candida albicans. To research the generation of the antibacterial phenomenon of LGT-5 and provide support for further research and application, the whole genome sequencing (WGS) of LGT-5 was obtained by single-molecule real-time DNA sequencing platform Pacific Biosciences (PacBio) sequencing and Illumina paired-end sequencing. The final assembled LGT-5 genome is 54.79 Mb with a contig N50 of 290.07 kb; in addition, its secondary metabolites were detected through HPLC-Q-ToF-MS/MS. By comparing its MS/MS data, the secondary metabolites were analyzed based on visual network maps obtained on the Global Natural Products Social Molecular Networking (GNPS). The analysis results showed that the secondary metabolites of LGT-5 were triterpenes and various cyclic dipeptides.

Structural insights into BCDX2 complex function in homologous recombination.

Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly byRAD51andsingle-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3-6 inactivate DNA binding and theHR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.

Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore.

This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a single-molecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

Plasmonic Bowl-Shaped Nanopore for Raman Detection of Single DNA Molecules in Flow-Through.

Plasmonic nanopores combined with Raman spectroscopy are emerging as platforms for single-molecule detection and sequencing in label-free mode. Recently, the ability of identifying single DNA bases or amino acids has been demonstrated for molecules adsorbed on plasmonic particles and then delivered into the plasmonic pores. Here, we report on bowl-shaped plasmonic gold nanopores capable of direct Raman detection of single λ-DNA molecules in a flow-through scheme. The bowl shape enables the incident laser to be focused into the nanopore to generate a single intense hot spot with no cut off in pore size. Therefore, we achieved ultrasmall focusing of NIR light in a spot of 3 nm. This enabled us to detect 7 consecutive bases along the DNA chain in flow-through conditions. Furthermore, we found a novel electrofluidic mechanism to manipulate the molecular trajectory within the pore volume so that the molecule is pushed toward the hot spot, thus improving the detection efficiency.

Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion.

The oncolytic autonomous parvovirus Minute Virus of Mice (MVM) establishes infection in the nuclear environment by usurping host DNA damage signaling proteins in the vicinity of cellular DNA break sites. MVM replication induces a global cellular DNA Damage Response (DDR) that is dependent on signaling by the ATM kinase and inactivates the cellular ATR-kinase pathway. However, the mechanism of how MVM generates cellular DNA breaks remains unknown. Using single molecule DNA Fiber Analysis, we have discovered that MVM infection leads to a shortening of host replication forks as infection progresses, as well as induction of replication stress prior to the initiation of virus replication. Ectopically expressed viral non-structural proteins NS1 and NS2 are sufficient to cause host-cell replication stress, as is the presence of UV-inactivated non-replicative MVM genomes. The host single-stranded DNA binding protein Replication Protein A (RPA) associates with the UV-inactivated MVM genomes, suggesting MVM genomes might serve as a sink for cellular stores of RPA. Overexpressing RPA in host cells prior to UV-MVM infection rescues DNA fiber lengths and increases MVM replication, confirming that MVM genomes deplete RPA stores to cause replication stress. Together, these results indicate that parvovirus genomes induce replication stress through RPA exhaustion, rendering the host genome vulnerable to additional DNA breaks.

An algorithm and application to efficiently analyse DNA fibre data

The duplication of genetic information (DNA replication) is central to life. Numerous control mechanisms ensure the exact course of the process during each cell division. Disturbances of DNA replication have severe consequences for the affected cell, and current models link them to cancer development. One of the most accurate methods for studying DNA replication is labelling newly synthesised DNA molecules with halogenated nucleotides, followed by immunofluorescence and microscopy detection, known as DNA fibre labelling. The method allows the registration of the activity of single replication complexes by measuring the length of the “trace” left by each of them. The major difficulty of the method is the labor-intensive analysis, which requires measuring the lengths of a large number of labelled fragments. Recently, the interest in this kind of image analysis has grown rapidly. In this project we have developed an algorithm and a lightweight Java application to automatically analyse single DNA molecule images that we have named “DNA size finder”. DNA size finder significantly simplified the analysis of the experimental data while increasing reliability by the standardised measurement of a greater number of DNA molecules. It is freely available and does not require any paid platforms or services to be used. We hope that the application will facilitate both the study of DNA replication control and the effects of various compounds used in human activity on the process of DNA replication.

Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA

Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (∼350 nucleotides per second) in the 5'→3' direction over relatively long distances (∼29,500 nucleotides). Surprisingly, we show the ssDNA-binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA.

Substantial Slowing of Electrophoretic Translocation of DNA through a Nanopore Using Coherent Multiple Entropic Traps.

One of the major challenges in the technology of sequencing DNA using single-molecule electrophoresis through a nanopore is to control the translocation of the macromolecule across the pore in order to allow sufficient time for accurate sequence reading at limited recording bandwidths. If the translocation speed is too fast, the signatures of the bases passing through the sensing region of the nanopore overlap in time, presenting difficulties in accurately identifying the bases in a sequential manner. Even though several strategies, such as enzyme ratcheting, have been implemented to reduce the translocation speed, the challenge to achieve a substantial reduction in the translocation speed continues to be of paramount significance. Toward achieving this goal, we have fabricated a nonenzymatic hybrid device that can reduce the translocation speed of long DNAs by more than 2 orders of magnitude, in comparison with the current status of the art. This device is made of a tetra-PEG hydrogel that is chemically anchored to the donor side of a solid-state nanopore. The idea behind this device is based on the recent discovery of the topologically frustrated dynamical state of confined polymers, whereby the front hydrogel matter of the hybrid device provides multiple entropic traps for a single DNA molecule holding it back against the electrophoretic driving force that pulls the DNA through the solid-state nanopore portion of the device. As a demonstration of slowing DNA translocation by a factor of about 500, we find the average translocation time realized in the present hybrid device for 3 kbp DNA as 23.4 ms, whereas the corresponding time for the bare solid-state nanopore under otherwise identical conditions is 0.047 ms. Our measurements on 1 kbp DNA and λ-DNA show that such a slowing down of DNA translocation with our hybrid device is general. An additional feature of our hybrid device is its incorporation of all features of the conventional gel electrophoresis to separate different DNA sizes in a clump of DNAs and to streamline them in an orderly and slow manner into the nanopore. Our results suggest the high potential of our hydrogel-nanopore hybrid device in further advancing the single-molecule electrophoresis technology to accurately sequence very large biological polymers.

Single duplex DNA sequencing with CODEC detects mutations with high sensitivity

Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules (‘single duplexes’) to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10−8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.

Abstract LB048: AR-targeted linked-read sequencing reveals complex AR structural variants in CRPC tumors

Abstract Inhibition of the androgen receptor (AR) transcription factor is the mainstay therapeutic strategy for systemic prostate cancer. However, development of a resistance phenotype termed castration resistant prostate cancer (CRPC) enables disease progression that underlies virtually all prostate cancer mortality. In the majority of CRPC, AR is transcriptionally re-activated in part due to accumulation of numerous alterations in the AR gene. These genomic alterations include missense mutations in exons that encode the ligand binding domain of AR, amplification of the AR gene body and/or an upstream transcriptional enhancer, and AR structural variants (SVs) resulting from inaccurate repair of DNA double strand breaks. In CRPC patient samples, these AR genomic alterations co-occur within tumors, which makes elucidating the fitness contribution of each alteration difficult. While short-read DNA sequencing has allowed for characterization of these events, they are underpowered to preserve linkage information. To address this challenge, we performed AR-targeted linked-read DNA-sequencing of CRPC cell line and patient derived xenograft (PDX) models with the goal of resolving sub-clonal and complex AR SVs. This approach confirmed the heterogeneity of AR SVs in these PDXs, including phased AR SVs that can be traced back to a single high molecular weight DNA molecule. These phased AR SVs were consistently found to co-occur in CRPC specimens with amplification of AR. Our results define a new class of AR SVs where multiple rearrangements co-occur on amplified DNA molecules to yield a complex array of AR gene structures in CRPC. Citation Format: Andrej Zivanovic, Sarah A. Munro, Jeffrey T. Miller, Yingming Li, Courtney N. Passow, Scott M. Dehm. AR-targeted linked-read sequencing reveals complex AR structural variants in CRPC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB048.

Monitoring the compaction of single DNA molecules in Xenopus egg extract in real time

DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed Xenopus egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.

Effect of Flow Rate and Ionic Strength on the Stabilities of YOYO-1 and YO-PRO-1 Intercalated in DNA Molecules.

Single-molecule DNA studies have improved our understanding of the DNAs' structure and their interactions with other molecules. A variety of DNA labeling dyes are available for single-molecule studies, among which the bis-intercalating dye YOYO-1 and mono-intercalating dye YO-PRO-1 are widely used. They have an extraordinarily strong affinity toward DNA and are bright with a high quantum yield (>0.5) when bound to DNAs. However, it is still not clear how these dyes behave in DNA molecules under higher ionic strength and strong buffer flow. Here, we have studied the effect of ionic strength and flow rate of buffer on their binding in single DNA molecules. The larger the flow rate and the higher the ionic strength, the faster the intercalated dyes are washed away from the DNAs. In the buffer with 1 M ionic strength, YOYO-1 and YO-PRO-1 are mostly washed away from DNA within 2 min of moderate buffer flow.

Driving Forces for Single DNA Stretching Assessed by In Situ TIRFM.

Herein, single DNA molecule stretching in a hydrodynamic flow was monitored by in situ total internal reflection fluorescence microscopy (TIRFM). Different driving forces for DNA stretching were assessed systematically. The binding force between the substrate and the DNA molecule significantly affected the dynamic stretching behavior of coiled DNA, where DNA stretched only on the substrate modified by -NH2 due to electrostatic adsorption. Proper flow force from the fluid was favorable for DNA stretching, which was in stark contrast to DNA stretching restricting at a lower flow rate or washing off DNA molecules at a higher flow rate of fluid. Moreover, DNA stretching was restricted in low pH solution but improved in high pH solution. This is due to the confrontation between contractive and repulsion forces in coiled DNA molecules in different pH, which is related to the Donnan equilibrium in a single condensed DNA molecule. Additionally, external Na+ would break this Donnan equilibrium, leading to a restriction for DNA stretching. This study will guide quantitative dynamics studies of DNA stretching in the future.

High-resolution single-molecule long-fragment rRNA gene amplicon sequencing of bacterial and eukaryotic microbial communities

Sequencing of hypervariable regions as well as internal transcribed spacer regions of ribosomal RNA genes (rDNA) is broadly used to identify bacteria and fungi, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length using high throughput, cost-efficient second-generation sequencing. We developed a method to obtain nearly full-length rDNA by assembling single DNA molecules combining DNA co-barcoding with single-tube long fragment read technology and second-generation sequencing. Benchmarking was performed using mock bacterial and fungal communities as well as two forest soil samples. All mock species rDNA were successfully recovered with identities above 99.5% compared to the reference sequences. From the soil samples we obtained good coverage with identification of more than 20,000 unknown species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.

Strain-level bacterial typing directly from patient samples using optical DNA mapping

BackgroundIdentification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.MethodsIn this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections.ResultsWe demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids.ConclusionThe optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management.