BACKGROUND: DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads.
RESULTS: We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12-15 h of sequencing.
CONCLUSIONS: Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.

Cattle produce Abs with an H chain ultralong CDR3 (40-70 aa). These Abs have been shown to have features such as broad neutralization of viruses and are investigated as human therapeutics. A common issue in sequencing the bovine BCR repertoire is the sequence length required to capture variable (V) and isotype gene information. This study aimed to assess the use of Oxford Nanopore Technologies\' MinION platform to perform IgM BCR repertoire sequencing to assess variation in the percentage of ultralong CDR3s among dairy cattle. Blood was collected from nine Holstein heifers. B cells were isolated using magnetic bead-based separation, RNA was extracted, and IgM+ transcripts were amplified using PCR and sequenced using a MinION R10.4 flow cell. The distribution of CDR3 lengths was trimodal, and the percentage of ultralong CDR3s ranged among animals from 2.32 to 20.13% in DNA sequences and 1.56% to 17.02% in productive protein sequences. V segment usage varied significantly among heifers. Segment IGHV1-7, associated with ultralong CDR3s, was used in 5.8-24.2% of sequences; usage was positively correlated with ultralong CDR3 production (r = 0.99, p < 0.01). To our knowledge, this is the first study to sequence the bovine BCR repertoire using Oxford Nanopore Technologies and demonstrates the potential for cost-efficient long-read repertoire sequencing in cattle without assembly. Findings from this study support literature describing the distribution of length and percentage of ultralong CDR3s. Future studies will investigate changes in the bovine BCR repertoire associated with age, antigenic exposure, and genetics.

Sageretia thea, a notable species within the mock buckthorn genus, is recognized for its intriguing biogeographical distribution and diverse medicinal properties. Despite this significance, genomic studies on S. thea are still in the nascent stages. We present the first chromosome-level genome assembly of S. thea that was generated using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies complemented by Pore-C chromatin conformation capture. The genome assembly had a size of 197.8 Mb with 12 chromosomal scaffolds and a scaffold N50 length of 15.9 Mb. A total of 25,434 protein-coding genes were identified and functionally annotated, and the gene model indicated 96.5% complete eukaryotic BUSCOs. Additionally, orthologous gene profiling and synteny analysis were performed to elucidate the evolutionary relationships within the Rhamnaceae family and Rosales. This high-quality chromosomal genome is the first genomic view of S. thea, which will serve as the basis for future studies on its biological and medicinal properties, and evolutionary history.

As an important biomarker, tumor cell-derived exosomes have substantial application prospects in early cancer screening and diagnosis. However, the unsatisfactory sensitivity and complicated sample pretreatment processes of conventional detection approaches have limited their use in clinical diagnosis. Nanopore sensors, as a highly sensitive, label-free, single-molecule technology, are widely utilized in molecule and bioparticle detection. Nevertheless, the exosome capture rate through nanopores is extremely low due to the low surface charge densities of exosomes and the effects of electrolyte concentration on their structural stability, thereby reducing the detection throughput. Here, we report an approach to improve the capture rate of exosome translocations using silicon nitride (SiNx) nanopores assisted by a slight salt electrolyte gradient. Improvements in exosome translocation event frequency are assessed in electrolyte solutions with different concentration gradients. In the case of asymmetric electrolytes (cis1× PBS and trans0.2 M NaCl, 1× PBS), the event frequency of tumor cell (HepG2)-derived exosome translocations is enhanced by nearly 2 orders of magnitude while maintaining vesicle structure stability. Furthermore, benefiting from the salt gradient effect, tumor cell (AsPC-1 and HCT116)-derived exosome translocations could be discriminated from those of HepG2 cell-derived exosomes. The developed highly sensitive detection method for tumor cell-derived exosomes at the single-particle level provides an approach for early cancer diagnosis.

This prospect explores the integration of enrichment strategies with nanopore detection to advance clinical glycoproteomics. Glycoproteins, crucial for understanding biological processes, pose challenges due to their low abundance and structural diversity. Enrichment techniques using lectin affinity, boronate affinity, and hydrazide chemistry and especially molecular imprinted polymers may selectively and specifically isolate glycoproteins from complex samples, while nanopore technology enables label-free, real-time, and single-molecule analysis. This approach holds promise for disease-related glycosylation studies, biomarker discovery, personalized medicine, and streamlined clinical analysis. Standardization, optimization, and data analysis remain challenges, requiring interdisciplinary collaborations and technological advancements. Overall, this integration may offer transformative potential for clinical glycoproteomics and innovative diagnostic and therapeutic strategies.

Electrospray ionization is widely used to generate vapor phase ions for analysis by mass spectrometry in proteomics research. However, only a small fraction of the analyte enters the mass spectrometer due to losses that are fundamentally linked to the use of a background gas to stimulate the generation of ions from electrosprayed droplets. Here we report a nanopore ion source that delivers ions directly into high vacuum from aqueous solutions. The ion source comprises a pulled quartz pipette with a sub-100 nm opening. Ions escape an electrified meniscus by ion evaporation and travel along collisionless trajectories to the ion detector. We measure mass spectra of 16 different amino acid ions, post-translationally modified variants of glutathione, and the peptide angiotensin II, showing that these analytes can be emitted as desolvated ions. The emitted current is composed of ions rather than charged droplets, and more than 90% of the current can be recovered in a distant collector.

Nanopore selective sequencing allows the targeted sequencing of DNA of interest using computational approaches rather than experimental methods such as targeted multiplex polymerase chain reaction or hybridization capture. Compared to sequence-alignment strategies, deep learning (DL) models for classifying target and nontarget DNA provide large speed advantages. However, the relatively low accuracy of these DL-based tools hinders their application in nanopore selective sequencing. Here, we present a DL-based tool named ReadCurrent for nanopore selective sequencing, which takes electric currents as inputs. ReadCurrent employs a modified very deep convolutional neural network (VDCNN) architecture, enabling significantly lower computational costs for training and quicker inference compared to conventional VDCNN. We evaluated the performance of ReadCurrent across 10 nanopore sequencing datasets spanning human, yeasts, bacteria, and viruses. We observed that ReadCurrent achieved a mean accuracy of 98.57% for classification, outperforming four other DL-based selective sequencing methods. In experimental validation that selectively sequenced microbial DNA from human DNA, ReadCurrent achieved an enrichment ratio of 2.85, which was higher than the 2.7 ratio achieved by MinKNOW using the sequence-alignment strategy. In summary, ReadCurrent can rapidly classify target and nontarget DNA with high accuracy, providing an alternative in the toolbox for nanopore selective sequencing. ReadCurrent is available at https://github.com/Ming-Ni-Group/ReadCurrent.

Saccharides, being one of the fundamental molecules of life, play essential roles in the physiological and pathological functions of cells. However, their intricate structures pose challenges for detection. Nanopore technology, with its high sensitivity and capability for single-molecule-level analysis, has revolutionized the identification and structural analysis of saccharide molecules. This review focuses on recent advancements in nanopore technology for carbohydrate detection, presenting an array of methods that leverage the molecular complexity of saccharides. Biological nanopore techniques utilize specific protein binding or pore modifications to trigger typical resistive pulses, enabling the high-sensitivity detection of monosaccharides and oligosaccharides. In solid-state nanopore sensing, boronic acid modification and pH gating mechanisms are employed for the specific recognition and quantitative analysis of polysaccharides. The integration of artificial intelligence algorithms can further enhance the accuracy and reliability of analyses. Serving as a crucial tool in carbohydrate detection, we foresee significant potential in the application of nanopore technology for the detection of carbohydrate molecules in disease diagnosis, drug screening, and biosensing, fostering innovative progress in related research domains.

The increasing prevalence of gut colonization with CTX-M extended-spectrum β-lactamase- and/or DHA plasmid-mediated AmpC-producing Escherichia coli is a concern. Here, we evaluate Nanopore-shotgun metagenomic sequencing (Nanopore-SMS) latest V14 chemistry to detect blaCTX-M and blaDHA genes from healthy stools. We test 25 paired samples characterized with culture-based methods (native and pre-enriched). Antimicrobial resistant genes (ARGs) are detected from reads and meta-assembled genomes (MAGs) to determine their associated genetic environments (AGEs). Sensitivity and specificity of native Nanopore-SMS are 61.1% and 100%, compared to 81.5% and 75% for pre-enriched Nanopore-SMS, respectively. Native Nanopore-SMS identifies only one sample with an AGE, whereas pre-enriched Nanopore-SMS recognizes 9/18 plasmids and 5/9 E. coli chromosomes. Pre-enriched Nanopore-SMS identifies more ARGs than native Nanopore-SMS (p < 0.001). Notably, blaCTX-Ms and blaDHAs AGEs (plasmid and chromosomes) are identified within 1 hour of sequencing. Furthermore, microbiota analyses show that pre-enriched Nanopore-SMS results in more E. coli classified reads (47% vs. 3.1%), higher differential abundance (5.69 log2 fold) and lower Shannon diversity index (p < 0.0001). Nanopore-SMS has the potential to be used for intestinal colonization screening. However, sample pre-enrichment is necessary to increase sensitivity. Further computational improvements are needed to reduce the turnaround time for clinical applications.

The present paper reported on the analysis of structural defects and their influence on the red-emitting γ-Al2O3:Mn4+,Mg2+ nanowires using positron annihilation spectroscopy (PAS). The nanowires were synthesized by hydrothermal method and low-temperature post-treatment using glucose as a reducing agent. X-ray diffraction (XRD), scanning electron microscopy (SEM), photoluminescence (PL), and photoluminescence excitation (PLE) were utilized, respectively, for determining the structural phase, morphology and red-emitting intensity in studied samples. Three PAS experiments, namely, positron annihilation lifetime (PAL), Doppler broadening (DB), and electron momentum distribution (EMD), were simultaneously performed to investigate the formations of structural defects in synthesized materials. Obtained results indicated that the doping concentration of 0.06% was optimal for the substitution of Mn4+ and Mg2+ to two Al3+ sites and the formation of oxygen vacancy (VO)-rich vacancy clusters (2VAl + 3VO) and large voids (~0.7 nm) with less Al atoms. Those characteristics reduced the energy transfer between Mn4+ ions, thus consequently enhanced the PL and PLE intensities. Moreover, this optimal doping concentration also effectively controlled the size of nanopores (~2.18 nm); hence, it is expected to maintain the high thermal conductivity of γ-Al2O3 nanowire-phosphor. The present study, therefore, demonstrated a potential application of γ-Al2O3 nanowire-phosphor in fabricating the high-performance optoelectronic devices.