Understanding pathogen transmission is key to effective infection prevention. From February to December 2021, we implemented genomics-enhanced contact tracing for SARS-CoV-2 in Düsseldorf, Germany, integrating data on 32,380 cases, 49,906 contact tracing records, 162 outbreaks, and 8,028 viral genomes (sequencing coverage 24.5%). Combining epidemiological and genetic data, we found a putative infection source for 19% of sequenced and 44% of all cases. Household-associated transmission accounted for up to 40% of all cases; genetic links for epidemiologically unexplained cases suggested limited sensitivity of classical contact tracing for non-household links. Outbreaks were associated with 8% of cases; school, kindergarten and nightlife outbreaks were strongly connected to the community, with nightlife outbreaks showing a strong post-outbreak increase in genetically associated cases. Sequencing detected previously unrecognized links between school outbreaks and 18% additional outbreak-associated sequenced cases. Across analyses, schools, kindergartens and gastronomy and nightlife settings were consistently identified as important contributors to transmission dynamics. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement We acknowledge funding by the Ministry for Work, Health and Social Affairs of the State of North Rhine-Westphalia ("24.04.01 Gen. u. IKA" and CPS-1-1A); the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung; Netzwerk Universitätsmedizin, GenSurv/MolTraX), award number 01KX2021; by the Jürgen Manchot Foundation; and by the Deutsche Forschungsgemeinschaft (DFG) award 428994620. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The ethics committee of the Medical Faculty of Heinrich Heine University Düsseldorf gave ethical approval for this work (#2020-839). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Code developed during the present study is available upon request to the authors.

Multiplexing in long-read DNA sequencing using Oxford Nanopore Technologies (ONT) increases throughput and reduces costs by ligating specific native barcodes to individual samples. However, this approach carries the risk of barcode misassignment (barcode crosstalk). Indeed, barcode crosstalk was observed after library preparation following the ONT standard protocol, particularly in samples with low DNA input. It is conceivable that misligation of residual native barcodes can occur during the subsequent sequencing adapter ligation step. To systematically investigate barcode crosstalk, genomic DNA of bacterial type strains with varying input concentrations was processed using three library preparation protocols: the former ONT standard protocol (protocol A, valid until July 2, 2025), an updated ONT protocol (protocol B), and an in-house protocol (protocol C) in which samples were pooled only after sequencing adapter ligation. Sequencing was performed with a Nanopore PromethIon. Protocol A showed substantial barcode crosstalk, especially in low-input samples, with up to 2.4% misassigned reads. Protocol B significantly reduced crosstalk to below 0.01%, likely due to improved washing conditions. Protocol C virtually eliminated crosstalk. These findings indicate that older ONT barcoding protocols should be critically reassessed. While the updated protocol improves accuracy, the in-house approach is recommended for low DNA input and high-precision sequencing.

Motivation In prokaryotic genomes, methylation is an important epigenetic modification that regulates the uptake of foreign DNA; it can also contribute to replication or virulence. We present MPore, a novel method for the database-driven detection of active methyltransferases and their associated target site recognition motifs from Nanopore R10 sequencing data of prokaryotic isolates. In contrast to existing methods, which typically start with the de novo identification of differentially methylated sequence motifs, MPore starts by identifying potential methyltransferase genes by homology search against REBASE; activity is then assessed through a regularized logistic regression model of observed genome-wide methylation patterns, integrating motif and genomic sequence context information.Results On two benchmarking datasets, 10 bacterial monocultures and two Helicobacter pylori genomes with complex methylation patterns, MPore achieved a combined recall of 93% and a combined PPV of 96%, outperforming Nanomotif (81%/91%), Modkit (66%/4%), and Snappy (89%/50%). Further validation on a well-characterized dataset of Mycoplasma hominis isolates showed perfect agreement with wet-lab-based validation results and demonstrated that MPore could complement REBASE information by disambiguating the specific methylated base in a motif with multiple potential methylation sites. MPore automatically produces integrated visualizations of the identified methyltransferases and observed methylation patterns; the tool is implemented as a user-friendly Snakemake pipeline.Availability and implementation MPore is freely available under the MIT license at https://github.com/DiltheyLab/MPore.

Abstract Multiplexing samples in long-read sequencing with Oxford Nanopore Next Generation Sequencing Technology (ONT) by ligating specific native barcodes to individual DNA samples enables significant increases of high throughput sequencing combined with a significant reduction of sequencing costs. However, this advantage carries the risk of barcode misassignment / crosstalk. Employing ONT multiplex sequencing with samples, we observed misassigned barcodes so called barcode crosstalk, after ONT library preparation according to the standard protocol, particularly in samples with low input DNA concentrations. We assumed that these barcode misassignments are largely due to misligation of remaining native barcodes during subsequent the subsequent sequencing adapter ligation. To systematically investigate and quantify barcode crosstalk, genomic DNA (gDNA) from four bacterial type strains with different DNA input concentrations was prepared using three protocols for library preparation: the Nanopore standard protocol (protocol A: version valid until July 2, 2025) the new Nanopore protocol (protocol B: version from July 2, 2025), and an in house protocol with pooling of the barcoded samples only after the sequencing adapter ligation step (protocol C: in house). All samples were sequenced on a Nanopore PromethIon device. The results clearly showed that the use of protocol A resulted in a pronounced barcode crosstalk especially detectable in samples with low DNA input concentrations (up to 2.4% misassigned reads). The ONT adjustment in protocol B (altered washing buffer vs. protocol A) significantly alleviated the barcode crosstalk to below 0.01%, whereas protocol C eliminated barcode crosstalk virtually completely. These observations emphasize that sequencing results obtained with older ONT native barcoding protocol variants should be critically reviewed. The newer ONT barcoding protocol is preferable for sequencing, but it does not completely eliminate the barcode crosstalk effect. In conclusion, for low DNA input and high accuracy sequencing, protocol C is recommended.

Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the host’s intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, to unravel mGBP2 functions, we report Galectin-9 (Gal9) as a critical mGBP2 interaction partner engaged for immunity to T. gondii. Interestingly, Gal9 also accumulates and colocalizes with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that Gal9 is a crucial factor for the mGBP2-coordinated cell-autonomous host defense mechanism against T. gondii.

Toxoplasma gondii (T. gondii) virulence in mice depends on different multiprotein complexes that assemble at the parasitophorous vacuole membrane (PVM) of the parasite. Individual rhoptry proteins within these complexes inhibit different Immunity-Related GTPases (IRG proteins). The rhoptry pseudokinase ROP5 is a central element to achieve IRG-specific rhoptry kinase activity and/or efficient complex formation. The rop5 locus of each of the canonical T. gondii strains encodes three major isoforms, ROP5A, ROP5B and ROP5C, and was shown to have the largest impact on virulence. By reverse genetics, we have generated T. gondii strains expressing either ROP5A, ROP5B or ROP5C in a RHΔrop5 genetic background and demonstrate that ROP5B is mainly responsible for heightened virulence of type I T. gondii in laboratory strains of mice. In vivo virulence correlates with diminished vacuolar IRG protein loading and parasite control in vitro only in presence of ROP5B but not ROP5A or ROP5C. Our results suggest that ROP5A and ROP5C isoforms might have co-evolved with IRG proteins or other host cell resistance factors in evolutionarily important intermediate hosts beyond Mus musculus. The same parasite effectors that inhibit IRG protein accumulation and function reduce the vacuolar amount of Guanylate Binding Proteins (GBP proteins). However, a parasite effector targeting a GBP protein at the PVM has not been described yet. Using two different approaches, Yeast Two-Hybrid analysis and Protein-fragment complementation assay, we here identified three heterologous IRG:GBP pairs, GBP6:Irgb10, GBP5:Irgb10, GBP5:Irgb6, and demonstrate that the accumulation of these GTPases at the PVM is interdependent. Our results offer a novel perspective on the IRG and GBP protein-mediated control of T. gondii infections and may further advance the investigation of GBP-specific T. gondii effectors.

Background/Objectives: Numerous intrinsic and extrinsic stressors can disrupt the balance of the skin microbiome, leading to the development of various skin diseases. It has been proven that coagulase-negative staphylococci (CoNS) are important commensals for maintaining skin microbiome homeostasis and fighting cutaneous pathogens such as Staphylococcus aureus (S. aureus). Here, we examined the influence of polyphenol-rich enzymatic blackcurrant extract (EBCE) on pathogenic coagulase-positive S. aureus strains and beneficial CoNS, like Staphylococcus epidermidis (S. epidermidis), to explore its potential for rebalancing the skin microbiota. Methods: The polyphenol profile of EBCE was determined by ultra-high-pressure liquid chromatography–tandem mass spectrometry. Microwell plate assays were employed to study the effect of EBCE on five S. aureus strains isolated from the skin of atopic dermatitis patients. An in vitro human stratum corneum model was used to test its effect on mixed bacterial cultures. Results: EBCE inhibited the growth of all tested S. aureus strains by 80–100% at the highest tested concentration after 7 h. No microbial growth was observed at the highest tested EBCE concentration using the stratum corneum model inoculated with one selected pathogen (S. aureus SA-DUS-017) and one commensal laboratory strain (S. epidermidis DSM 20044). The lowest tested concentration did not interfere with S. aureus growth but strongly stimulated the growth of S. epidermidis (~300-fold colony forming unit increase). In addition, low EBCE concentrations strongly stimulated CoNS growth in microbiome samples taken from the armpits of healthy volunteers that were spiked with S. aureus SA-DUS-017. Conclusions: These preclinical data support further testing of EBCE-enriched topical preparations as potential cutaneous prebiotics in human studies.

Lymphotoxin β receptor (LTβR/TNFRSF3) signaling plays a crucial role in immune defense. Notably, LTβR-deficient (LTβR-/-) mice exhibit severe defects in innate and adaptive immunity against various pathogens and succumb to Toxoplasma gondii infection. Here, we investigated the bone marrow (BM) and peritoneal cavity (PerC) compartments of LTβR-/- mice during T. gondii infection, demonstrating perturbed B-cell and T-cell subpopulations in the absence of LTβR signaling. T. gondii infection disrupted BM lymphopoiesis, depleting early and mature B cells in WT mice, whereas mature B cells remained present in LTβR-/- BM. LTβR-/- BM also exhibited reduced MHCII+ monocytes and a plasma cell compartment skewed toward IgM+ rather than IgA+ cells. In addition, BM Tcell subsets were altered, exhibiting decreased double-negative (CD4-/CD8-) and increased CD4+ and CD8+ T-cell frequencies. Analysis of the BM transcriptome revealed diminished interferon responses but an upregulated TNFα-NF-κB signaling signature in uninfected and infected LTβR-/- mice, potentially compensating for the absence of LTβR signaling. LTβR-/- mice displayed an altered B-1a to B-1b ratio and a predominant presence of neutrophils in the PerC. In summary, we identified novel immunological alterations in the BM and PerC compartments of LTβR-/- mice, which suggest new roles for LTβR signaling in B- and T-cell homeostasis, migration, and pathogen defense.

Secondary bacterial infection, often caused by Streptococcus pneumoniae, is one of the most frequent and severe complications of influenza A virus-induced (IAV-induced) pneumonia. Phenotyping of the pulmonary immune cell landscape after IAV infection revealed a substantial depletion of the tissue-resident alveolar macrophage (TR-AM) population at day 7, which was associated with increased susceptibility to S. pneumoniae outgrowth. To elucidate the molecular mechanisms underlying TR-AM depletion, and to define putative targets for treatment, we combined single-cell transcriptomics and cell-specific PCR profiling in an unbiased manner, using in vivo models of IAV infection and IAV and S. pneumoniae coinfection. The TNF superfamily 14 (TNFSF14) ligand/receptor axis was revealed as the driving force behind post-influenza TR-AM death during the early infection phase, enabling the transition to pneumococcal pneumonia, whereas intrapulmonary transfer of genetically modified TR-AMs and antibody-mediated neutralization of specific pathway components alleviated disease severity. With mainly neutrophilic expression and high abundance in the bronchoalveolar fluid of patients with severe virus-induced acute respiratory distress syndrome, TNFSF14 emerged as a key determinant of virus-driven lung injury. Targeting the TNFSF14-mediated intercellular communication network in the virus-infected lung can, therefore, improve host defense, minimizing the risk of subsequent bacterial pneumonia and ameliorating the disease outcome.

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The heterogeneity of conventional dendritic cells type 1 (cDC1s) and type 2 (cDC2s) is well established, yet the identity and origin of CD301b+ cDC2s remain debated. Here, we show that CD301b+ cDC2s and CD103+ cDC1s develop from pre-committed progenitors in response to granulocyte/macrophage colony-stimulating factor (GM-CSF). While CD103+ cDC1s acquire their phenotype and functional properties through GM-CSF-driven differentiation from pre-cDC1s, CD301b+ cDC2s emerge as cytokine-induced states from DC2- and DC3-committed progenitors. CD103+ cDC1s and CD301b+ cDC2s exhibit enhanced T cell priming capacities and distinct cytokine expression profiles upon GM-CSF exposure. In vivo, DC-intrinsic GM-CSF sensing is dispensable for acquiring CD103 and CD301b expression with the notable exception of lung DCs, while specific type 2 cytokines induce CD103 and CD301b ex vivo. These findings identify GM-CSF and specific type 2 cytokines as central regulators of cDC1 and cDC2 effector differentiation and establish CD301b as a marker of a cytokine-driven cDC2 state.

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-β-thia and β-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.

The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members is limited using 16S rDNA sequencing. Here, we developed a pipeline enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using our pipeline we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, characterized by Bacteroides spp. /Phocaeicola spp., mixed composition and Enterococcus abundances. We revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples. In the course of HSCT, bacterial strains were stable or newly acquired. Our results demonstrate the disruptive potential of alloHSCTon the gut microbiome and pave the way for future comprehensive microbiome studies based on long-read metagenomics.

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum. Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar PfDXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N-phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N-terminal domain. As shown for reverse carba and thia analogs, PfDXR selectively binds the S-enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.

Background/Objectives: Patient-specific diagnostic and therapeutic approaches are important in the care of people with chronic wounds. The heterogeneity of underlying disease profiles and the diversity of the wound micro-environment make generalized approaches difficult. While high-throughput molecular diagnostic methods are increasingly widespread and available, the analysis of objective biomolecular disease patterns has not found its way into everyday wound management. The aim of this study is to evaluate the use of wound swab samples for the analysis of biomarkers and disease patterns in people with chronic wounds. Methods: A sample cohort from the multicenter “Wound-BIOME” project was analyzed. The project aims to comprehensively investigate the local micro-environment of chronic wounds of various entities, healing tendencies and regeneration stages at the biomolecular level. A sample collection and handling protocol suitable for everyday use was tested and evaluated regarding feasibility for multiplex immunoassay, proteomics, small RNA sequencing (miRNA) and metagenome analyses (microbiomics). Results: It could be shown that standard wound swabs are well-suited for the analysis of the complex wound micro-environment using various high-throughput methods. Despite the sample heterogeneity, the quality was adequate to analyze biomolecular patterns. Conclusions: Initial analyses of protein signatures, microbial wound communities and miRNA patterns show promising results for future individualized diagnostics and targeted interventions.

Background/Objectives: In oral and maxillofacial surgery, the reconstruction of defects often involves the transfer of skin tissue into the oral cavity utilizing microvascular grafts. This study investigates postoperative changes in microbial colonization following intraoral microvascular transplantation, as well as potential influencing factors. Methods: In 37 patients undergoing intraoral reconstructions, pre- and postoperative swabs were taken from the donor and recipient regions to quantify the seven selected marker bacteria using TaqMan PCRs. Patient-specific factors and clinical data were also recorded. Results: The infection-associated Acinetobacter baumannii tended to decrease postoperatively, while the infectious pathogens Pseudomonas aeruginosa, Enterococcus faecalis and the family of Enterobacteriaceae showed a postoperative increase without being directly associated with a clinical infection. Streptococcus mitis showed a significant postoperative decrease on buccal mucosa and increase on the graft surface (oral dysbiosis) and was significantly reduced or displaced by other bacteria (e.g., Mycoplasma salivarium, positive selection) when treated with ampicillin/sulbactam. Conclusions: The cutaneous microbiome of the graft adapts to the local intraoral environment. Postoperative shifts in oral bacterial colonization and an increase in infection-relevant bacteria were observed. These perioperative changes in colonization are also influenced by the administration of ampicillin/sulbactam. Consequently, single doses of antibiotics appear to be more beneficial compared to longer-term preventive use.

(1) Background: Microbial keratitis is a serious eye infection that carries a significant risk of vision loss. Acanthamoeba spp. are known to cause keratitis and their bacterial endosymbionts can increase virulence and/or treatment resistance and thus significantly worsen the course of the disease. (2) Methods and Results: In a suspected case of Acanthamoeba keratitis, in addition to Acanthamoeba spp., an endosymbiont of acanthamoebae belonging to the taxonomic order of Holosporales was detected by chance in a bacterial 16S rDNA-based pan-PCR and subsequently classified as Candidatus Paracaedibacter symbiosus through an analysis of an enlarged 16S rDNA region. We used Oxford Nanopore Technology to evaluate the usefulness of whole-genome sequencing (WGS) as a one-step diagnostics method. Here, Acanthamoeba castellanii and the endosymbiont Candidatus Paracaedibacter symbiosus could be directly detected at the species level. No other microbes were identified in the specimen. (3) Conclusions: We recommend the introduction of WGS as a diagnostic approach for keratitis to replace the need for multiple species-specific qPCRs in future routine diagnostics and to enable an all-encompassing characterisation of the polymicrobial community in one step.

Genomic surveillance enables the early detection of pathogen transmission in healthcare facilities and contributes to the reduction of substantial patient harm. Fast turnaround times, flexible multiplexing, and low capital requirements make Nanopore sequencing well suited for genomic surveillance purposes; the analysis of Nanopore data, however, can be challenging. We present NanoCore, a user-friendly method for Nanopore-based genomic surveillance in healthcare facilities, enabling the calculation and visualization of cgMLST-like (core-genome multilocus sequence typing) sample distances directly from unassembled Nanopore reads. NanoCore implements a mapping, variant calling, and multilevel filtering strategy and also supports the analysis of Illumina data. We validated NanoCore on two 24-isolate data sets of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). In the Nanopore-only mode, NanoCore-based pairwise distances between closely related isolates were near-identical to Illumina-based SeqSphere+ distances, a gold standard commercial method (average differences of 0.75 and 0.81 alleles for MRSA and VRE; sd = 0.98 and 1.00), and gave an identical clustering into closely related and non-closely related isolates. In the "hybrid" mode, in which only Nanopore data are used for some isolates and only Illumina data for others, increased average pairwise isolate distance differences were observed (average differences of 3.44 and 1.95 for MRSA and VRE, respectively; sd = 2.76 and 1.34), while clustering results remained identical. NanoCore is computationally efficient (<15 hours of wall time for the analysis of a 24-isolate data set on a workstation), available as free software, and supports installation via conda. In conclusion, NanoCore enables the effective use of the Nanopore technology for bacterial pathogen surveillance in healthcare facilities. IMPORTANCE:Genomic surveillance involves sequencing the genomes and measuring the relatedness of bacteria from different patients or locations in the same healthcare facility, enabling an improved understanding of pathogen transmission pathways and the detection of "silent" outbreaks that would otherwise go undetected. It has become an indispensable tool for the detection and prevention of healthcare-associated infections and is routinely applied by many healthcare institutions. The earlier an outbreak or transmission chain is detected, the better; in this context, the Oxford Nanopore sequencing technology has important potential advantages over traditionally used short-read sequencing technologies, because it supports "real-time" data generation and the cost-effective "on demand" sequencing of small numbers of bacterial isolates. The analysis of Nanopore sequencing data, however, can be challenging. We present NanoCore, a user-friendly software for genomic surveillance that works directly based on Nanopore sequencing reads in FASTQ format, and demonstrate that its accuracy is equivalent to traditional gold standard short read-based analyses.