Rapid Barcoding Research Articles

Portable Food-Freshness Prediction Platform Based on Colorimetric Barcode Combinatorics and Deep Convolutional Neural Networks.

Artificial scent screening systems (known as electronic noses, E-noses) have been researched extensively. A portable, automatic, and accurate, real-time E-nose requires both robust cross-reactive sensing and fingerprint pattern recognition. Few E-noses have been commercialized because they suffer from either sensing or pattern-recognition issues. Here, cross-reactive colorimetric barcode combinatorics and deep convolutional neural networks (DCNNs) are combined to form a system for monitoring meat freshness that concurrently provides scent fingerprint and fingerprint recognition. The barcodes-comprising 20 different types of porous nanocomposites of chitosan, dye, and cellulose acetate-form scent fingerprints that are identifiable by DCNN. A fully supervised DCNN trained using 3475 labeled barcode images predicts meat freshness with an overall accuracy of 98.5%. Incorporating DCNN into a smartphone application forms a simple platform for rapid barcode scanning and identification of food freshness in real time. The system is fast, accurate, and non-destructive, enabling consumers and all stakeholders in the food supply chain to monitor food freshness.

Rapid and Accurate Species Identification of Mitis Group Streptococci Using the MinION Nanopore Sequencer.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner “What's in My Pot?” (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding.

Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

238. Direct identification of Bacterial Species with MinION Nanopore Sequencer In Clinical Specimens Suspected of Polybacterial Infection

BackgroundConventional culture tests usually identify only a few bacterial species, which can grow well in the culture system, in the cases of polybacterial infection. 16S rRNA gene nanopore sequencing enables semi-quantitative identification of bacterial genetic materials. We aimed to evaluate usefulness of 16s rRNA gene nanopore sequencing in the cases suspected of polybacterial infection.MethodsThe research was conducted in a single university hospital for one year. Conventional bacterial culture identification and nanopore sequencing of 16s rRNA gene were carried out simultaneously for cases where polybacterial infection is strongly suspected. Blood agar plate was used for conventional culture, and Microscan (Beckman Coulter, United States) and Vitek 2 (Biomerieux, FR) automated systems were used for identification. For nanopore sequencing, 16S rRNA gene PCR was performed from the clinical specimens, and sequencing libraries were generated from the PCR products using the rapid barcoding sequencing kit (Oxford nanopore technologies, UK). MinION sequencing was performed for 1–3 hours and the generated reads were analyzed using the EPI2ME 16S BLAST workflow.ResultsSpecimens were obtained from 15 patients; 6 liver abscess, 2 psoas abscess, 2 thigh abcess, 1 paraspinal abscess, 1 mycotic aneurysm, 1 necrotizing fasciitis, 1 fingertip gangrene and 1 abscess in coccyx area. 16s rRNA gene nanopore sequencing showed monobacterial organism in 8 (53.3%) specimens and polybacterial organisms in 7 (46.6%) specimens. In three (37.5%) cases of 8 cases with monobacterial infections identified by 16s rRNA gene sequencing, no organism was grown in conventional culture, possibly due to previous antibiotic administration. Notably, among 8 cases with polybacterial infection by 16s rRNA gene nanopore sequencing test, traditional culture test showed polybacterial infection in only two (25%) cases and single bacterial organism was identified in the other 6 (75%) cases.ConclusionNanopore sequencing of 16s rRNA gene using the MinION sequencer may be useful for identification of causing microorganism and differentiation between monobacterial and polybacterial infection when polybacterial infection is suspected.Disclosures All authors: No reported disclosures.

Efficient generation of complete sequences of MDR-encoding plasmids by rapid assembly of MinION barcoding sequencing data.

BackgroundMultidrug resistance (MDR)–encoding plasmids are considered major molecular vehicles responsible for transmission of antibiotic resistance genes among bacteria of the same or different species. Delineating the complete sequences of such plasmids could provide valuable insight into the evolution and transmission mechanisms underlying bacterial antibiotic resistance development. However, due to the presence of multiple repeats of mobile elements, complete sequencing of MDR plasmids remains technically complicated, expensive, and time-consuming.ResultsHere, we demonstrate a rapid and efficient approach to obtaining multiple MDR plasmid sequences through the use of the MinION nanopore sequencing platform, which is incorporated in a portable device. By assembling the long sequencing reads generated by a single MinION run according to a rapid barcoding sequencing protocol, we obtained the complete sequences of 20 plasmids harbored by multiple bacterial strains. Importantly, single long reads covering a plasmid end-to-end were recorded, indicating that de novo assembly may be unnecessary if the single reads exhibit high accuracy.ConclusionsThis workflow represents a convenient and cost-effective approach for systematic assessment of MDR plasmids responsible for treatment failure of bacterial infections, offering the opportunity to perform detailed molecular epidemiological studies to probe the evolutionary and transmission mechanisms of MDR-encoding elements.

Rapid diagnostic barcode system for codetection of multiple protein markers

An ultrasensitive immunodiagnostic readout method based on an electrochemical analysis is presented. Different inorganic quantum dot (QD) nanocrystals (ZnS, CdS, and PbS) are tagged to antibodies for the on-site voltammetric stripping measurements of multiple antigen targets. The multiprotein electrical sensing capability is coupled to the amplification feature of anodic stripping voltammetric transduction and with an efficient magnetic removal (to minimize nonspecific adsorption and cross-reactivity effects). Sandwich-immunoassay formats were performed using model proteins (/spl beta//sub 2/-microglobulin, myoglobin, and human serum albumin). These encoding QD tracers with distinct redox potential yield highly sensitive and selective stripping peaks at -1.11 V (Zn), -0.67 V (Cd), and -0.52 V (Pb) at the mercury-film screen printed carbon electrode (versus Ag/AgCl reference). The position and size of these peaks reflect the identity and risk level of the corresponding antigen marker. The favorable signal-to-noise characteristics of the response for the initial 25-ng/mL mixture indicate a detection limit of ca. 10 ng/mL far below the early warning range and allow a reliable determination of very low protein concentrations. Such analog peaks of the QDs were converted to simple and rapid barcode signals. The digital readout system can code 215 electrically tuned barcodes to mark different protein analytes and to be useful for a wireless communication system.