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Premise Target sequence capture (Hyb‐Seq) is a cost‐effective sequencing strategy that employs RNA probes to enrich for specific genomic sequences. By targeting conserved low‐copy orthologs, Hyb‐Seq enables efficient phylogenomic investigations. Here, we present Asparagaceae1726—a Hyb‐Seq probe set targeting 1726 low‐copy nuclear genes for phylogenomics in the angiosperm family Asparagaceae—which will aid the often‐challenging delineation and resolution of evolutionary relationships within Asparagaceae. Methods Here we describe and validate the Asparagaceae1726 probe set (https://github.com/bentzpc/Asparagaceae1726) in six of the seven subfamilies of Asparagaceae. We perform phylogenomic analyses with these 1726 loci and evaluate how inclusion of paralogs and bycatch plastome sequences can enhance phylogenomic inference with target‐enriched data sets. Results We recovered at least 82% of target orthologs from all sampled taxa, and phylogenomic analyses resulted in strong support for all subfamilial relationships. Additionally, topology and branch support were congruent between analyses with and without inclusion of target paralogs, suggesting that paralogs had limited effect on phylogenomic inference. Discussion Asparagaceae1726 is effective across the family and enables the generation of robust data sets for phylogenomics of any Asparagaceae taxon. Asparagaceae1726 establishes a standardized set of loci for phylogenomic analysis in Asparagaceae, which we hope will be widely used for extensible and reproducible investigations of diversification in the family.

The application of next generation sequencing (NGS) for the analysis of mitochondrial (mt) DNA, short tandem repeats (STRs), and single nucleotide polymorphism (SNPs) has demonstrated great promise for challenging forensic specimens, such as degraded, limited, and mixed samples. Target enrichment using probe capture rather than PCR amplification offers advantages for analysis of degraded DNA since two intact PCR primer sites in the template DNA molecule are not required. Furthermore, NGS software programs can help remove PCR duplicates to determine initial template copy numbers of a shotgun library. Moreover, the same shotgun library prepared from a limited DNA source can be enriched for mtDNA as well as nuclear markers by hybrid capture with the relevant probe panels. Here, we demonstrate the use of this strategy in the analysis of limited and mock degraded samples using our custom probe capture panels for massively parallel sequencing of the whole mtgenome and 426 SNP markers. We also applied the mtgenome capture panel in a mixed sample and analyzed using both phylogenetic and variant frequency based bioinformatics tools to resolve the minor and major contributors. Finally, the results obtained on individual telogen hairs demonstrate the potential of probe capture NGS analysis for both mtDNA and nuclear SNPs for challenging forensic specimens.

Since the invention of the first biosensors 70 years ago, they have turned into valuable and versatile tools for various applications, ranging from disease diagnosis to environmental monitoring. Traditionally, antibodies have been employed as the capture probes in most biosensors, owing to their innate ability to bind their target with high affinity and specificity, and are still considered as the gold standard. Yet, the resulting immunosensors often suffer from considerable limitations, which are mainly ascribed to the antibody size, conjugation chemistry, stability, and costs. Over the past decade, aptamers have emerged as promising alternative capture probes presenting some advantages over existing constraints of immunosensors, as well as new biosensing concepts. Herein, we review the employment of antibodies and aptamers as capture probes in biosensing platforms, addressing the main aspects of biosensor design and mechanism. We also aim to compare both capture probe classes from theoretical and experimental perspectives. Yet, we highlight that such comparisons are not straightforward, and these two families of capture probes should not be necessarily perceived as competing but rather as complementary. We, thus, elaborate on their combined use in hybrid biosensing schemes benefiting from the advantages of each biorecognition element.

As sequencing technology transitions from research to clinical settings, due to technological maturity and cost reductions, metagenomic next-generation sequencing (mNGS) is increasingly used. This shift underscores the growing need for more cost-effective and universally accessible sequencing assays to improve patient care and public health. Therefore, targeted NGS (tNGS) is gaining prominence. tNGS involves enrichment of target pathogens in patient samples based on multiplex PCR amplification or probe capture with excellent sensitivity. It is increasingly used in clinical diagnostics due to its practicality and efficiency. The present review compares the principles of different enrichment methods. The high positivity rate of tNGS in the detection of pathogens was found in respiratory samples with specific instances. tNGS maintains high sensitivity (70.8-95.0%) in samples with low pathogen loads, including blood and cerebrospinal fluid. Furthermore, tNGS is effective in detecting drug-resistant strains of Mycobacterium tuberculosis, allowing identification of resistance genes and guiding clinical treatment decisions, which is difficult to achieve with mNGS. In the present review, the application of tNGS in clinical settings and its current limitations are assessed. The continued development of tNGS has the potential to refine diagnostic accuracy and treatment efficacy and improving infectious disease management. However, further research to overcome technical challenges such as workflow time and cost is required.

Abstract At present, no single workflow is available for quick and accurate identification and analysis of genomes of various viruses present together in a field or clinical sample, particularly when followed by long-read sequencing. Our work addressed this limitation by combining targeted enrichment with long-read, real-time sequencing. Using a panel of probes targeting 16,069 complete viral genomes, we validated this workflow (termed TELSVirus) on complex sample matrices collected from pigs and compared its performance to traditional methods including real-time reverse transcription polymerase chain reaction and shotgun metagenomics. Using serial dilutions of samples with known viral status, we observed that TELSVirus generated viral reads for dilutions up to 10−9. TELSVirus was able to detect viral targets when shotgun metagenomic long- and short-read datasets did not and when rRT-PCR results were undetermined. Finally, we performed TELSVirus on 144 oral fluid samples collected in the field, which are highly complex and diverse samples used for viral surveillance in swine. We identified a high prevalence of relatively understudied viruses, often found co-circulating with better-characterized viruses. In many cases, TELSVirus generated ultra-deep genome coverage, allowing for further genomic epidemiological investigations, although bioinformatic methods need further development to work robustly with TELSVirus data. Our results support using TELSVirus for rapid detection and genomic characterization of multiple low-abundance viruses from single samples using long-read sequencing.

Background Sequencing viruses in many specimens is hindered by excessive background material from hosts, microbiota, and environmental organisms. Consequently, enrichment of target genomic material is necessary for practical high-throughput viral genome sequencing. Hybridization probes are widely used for enrichment in many fields, but their application to viral sequencing faces a major obstacle: it is difficult to design panels of probe oligo sequences that broadly target many viral taxa due to their rapid evolution, extensive diversity, and genetic hypervariability. To address this challenge, we created ProbeTools, a package of bioinformatic tools for generating effective viral capture panels, and for assessing coverage of target sequences by probe panel designs in silico . In this study, we validated ProbeTools by designing a panel of 3600 probes for subtyping the hypervariable haemagglutinin (HA) and neuraminidase (NA) genome segments of avian-origin influenza A viruses (AIVs). Using in silico assessment of AIV reference sequences and in vitro capture on egg-cultured viral isolates, we demonstrated effective performance by our custom AIV panel and ProbeTools’ suitability for challenging viral probe design applications. Results Based on ProbeTool’s in silico analysis, our panel provided broadly inclusive coverage of 14,772 HA and 11,967 NA reference sequences. For each reference sequence, we calculated the percentage of nucleotide positions covered by our panel in silico; 90% of HA and NA references sequences had at least 90.8 and 95.1% of their nucleotide positions covered respectively. We also observed effective in vitro capture on a representative collection of 23 egg-cultured AIVs that included isolates from wild birds, poultry, and humans and representatives from all HA and NA subtypes. Forty-two of forty-six HA and NA segments had over 98.3% of their nucleotide positions significantly enriched by our custom panel. These in vitro results were further used to validate ProbeTools’ in silico coverage assessment algorithm; 89.2% of in silico predictions were concordant with in vitro results. Conclusions ProbeTools generated an effective panel for subtyping AIVs that can be deployed for genomic surveillance, outbreak prevention, and pandemic preparedness. Effective probe design against hypervariable AIV targets also validated ProbeTools’ design and coverage assessment algorithms, demonstrating their suitability for other challenging viral capture applications.

The authors describe an electrochemical assay for fast detection of Escherichia coli (E. coli) . It is based on a dual signal amplification strategy and the use of a screen-printed carbon electrode (SPCE) whose surface was modified with a polyaniline (PANI) film and gold nanoparticles (AuNPs) via cyclic voltammetry (CV). In the next step, avidin was covalently immobilized on the PANI/AuNP composite on the SPCE surface. Subsequently, the biotinylated DNA capture probe was immobilized onto the PANI/AuNP/avidin-modified SPCE by biotin-avidin interaction. Then, DNA of E.coli , digoxigenin-labeled DNA detector probe and anti-digoxigenin-labeled horseradish peroxidase (HRP) were placed on the electrode. 3,3′,5,5′-Tetramethylbenzidine (TMB) and H 2 O 2 solution were added and the CV electrochemical signal was generated at a potential of −0.1 V (vs. Ag/AgCl) and a scan rate 50 mV.s −1 . The assay can detect 4 × 10 6 to 4 CFU of E. coli without DNA amplification. The biosensor is highly specific over other pathogens including Klebsiella pneumoniae , Proteus mirabilis , Enterococcus faecalis , Staphylococcus haemolyticus and Pseudomonas aeruginosa . It can be concluded that this genosensor has an excellent potential for rapid and accurate diagnosis of E.coli inflicted infections. Graphical Abstract Schematic of an electrochemical E. coli  genosensor based on sandwich assay on a polyaniline/gold nanoparticle-modified screen printed carbon electrode (SPCE). The biosensor can detect 4 × 10 6 to 4 CFU of E. coli without DNA amplification.

Purpose Targeted next-generation sequencing (tNGS) has emerged as an advanced diagnostic technique. While tNGS is increasingly recognized as a valuable tool for detecting infections, its most relevant clinical indications remain underdefined. This study aimed to evaluate the clinical utility of tNGS for lower respiratory tract infections (LRTIs). Methods We conducted a prospective, observational study to evaluate the clinical diagnostic value of broad-spectrum targeted Next-Generation Sequencing (bstNGS) covering 1872 microorganisms in critically ill patients with LRTIs. We compared the microbial detection performance of bstNGS, mNGS, and traditional culture methods in bronchoalveolar lavage fluid (BALF). Additionally, we used the odds ratio (OR) from multiple logistic regression to assess the impact of relevant clinical variables on the detection of pathogens by bstNGS. We also examined the correlation between bstNGS pathogen detection results and clinical outcomes. Results Between August 23, 2023, and April 24, 2024, samples from 150 patients were analyzed. bstNGS detected 96.33% and 91.15% of the microorganisms discovered by mNGS and culture respectively, and was capable of identifying microorganisms with even lower loads. According to the diagnostic criteria, bstNGS, mNGS, and culture methods detected pathogens in 87.33%, 82.00%, and 46.00% of the samples respectively. Moreover, the NGS methods demonstrated a stronger pathogen detection ability compared to culture ( p  < 0.05). Further comparing the diagnostic performance of the three methods, bstNGS exhibited higher diagnostic accuracy than both mNGS (90.67% vs 86.00%, p  < 0.05) and culture (90.67% vs 49.33%, p  < 0.0001). Multivariate analysis revealed that immunocompromise was associated with a lower efficiency of pathogen detection by bstNGS ( p  = 0.04), while other included clinical features had no significant correlation with bstNGS detection. Additionally, compared with patients in whom no pathogen was detected, patients in whom a pathogen was detected by bstNGS were associated with better outcomes of antibiotic treatment (89.68% vs. 62.50%; OR 7.53, 95% CI 1.41–45.30; p  = 0.02). Conclusion This study shows the effectiveness of bstNGS in detecting pathogens of LRTIs, as well as its value as a potential auxiliary diagnostic method in the ICU.

Abscisic acid (ABA), as the most common plant hormone in the growth of wheat, can greatly affect the yield when its levels deviate from normal. Therefore, highly sensitive and selective detection of this hormone is greatly needed. In this work, we developed an aptamer sensor based on surface-enhanced Raman spectroscopy (SERS) and applied it for the high sensitivity detection of ABA. Biotin-modified ABA aptamer complement chains were modified on ferrosoferric oxide magnetic nanoparticles (Fe3O4MNPs) and acted as capture probes, and sulfhydryl aptamer (SH-Apt)-modified silver-coated gold nanospheres (Au@Ag NPs) were used as signal probes. Through the recognition of the ABA aptamer and its complementary chains, an aptamer sensor based on SERS was constructed. As SERS internal standard molecules of 4-mercaptobenzoic acid (4-MBA) were encapsulated between the gold core and silver shell of the signal probes; the constructed aptamer sensor generated a strong SERS signal of 4-MBA after magnetic separation. When there were ABA molecules in the detection system, with the preferential binding of ABA aptamer and ABA molecule, the signal probes were released from the capture probes, after magnetic separation, leading to a linear decrease in SERS intensity of 4-MBA. Thus, the detection response was linear over a logarithmic concentration range, with an ultra-low detection limit of 0.67 fM. In addition, the practical use of this assay method was demonstrated in ABA detection from fresh wheat leaves, with a relative error (RE) of 5.43–8.94% when compared with results from enzyme-linked immunosorbent assay (ELISA). The low RE value proves that the aptamer sensor will be a promising method for ABA detection.

Background The clinical value of shotgun metagenomic next-generation sequencing (mNGS) in improving the detection rates of respiratory pathogens is well-established. However, mNGS is complex and expensive. This study designed and evaluated the performance of targeted NGS (tNGS) in diagnosing respiratory infections. Methods We retrospectively included samples from 281 patients with lower respiratory tract infections to establish thresholds of pathogens. Subsequently, target pathogens were selected and a probe hybridization system was established. The performance and clinical manifestations of tNGS for 306 pathogens were evaluated using clinical and simulated samples. Results The tNGS method took 16 h with sequencing data sizes of 5 M reads. The limit-of-detection of tNGS was 100–200 CFU/mL, respectively. Bioinformatics simulation confirmed the method’s high specificity and robustness. In 281 patients of clinical validation cohort, tNGS exhibited a sensitivity of 97.73% and specificity of 75.41% compared to the composite reference standard, which notably surpasses those of culture-based and conventional microbiological methods (CMT). In detecting bacterial and viral infection, tNGS demonstrated superior sensitivity relative to CMT. Notably, 61.40% of target viruses were subtype-resolved with the initial establishment of reliable typing cutoffs, with the subtyping results being completely consistent with the PCR results. tNGS allowed for concurrent identification of antimicrobial resistance (AMR) markers and viral subtyping. 80.56% of AMR markers identified by tNGS were consistent with antimicrobial susceptibility testing. Conclusion This research established the robust performance of our tailored tNGS assay in the simultaneous detection of DNA and RNA pathogens, underscoring its prospective suitability for widespread use in clinical diagnostics.