Pilot validation of on-field STR typing and human identity testing by MinION nanopore sequencing.

Nanopore sequencing technology has broad application prospects in forensic medicine due to its small size, portability, fast speed, real-time result analysis capabilities, single-molecule sequencing abilities, and simple operation. Here, we demonstrate for the first time that nanopore sequencing platforms can be used to identify individuals in the field. Through scientific and reasonable design, a nanopore MinION MK1B device and other auxiliary devices are integrated into a portable detection box conducive to individual identification at the accident site. Individual identification of 12 samples could be completed within approximately 24 h by jointly detecting 23 short tandem repeat (STR) loci. Through double-blinded experiments, the genotypes of 49 samples were successfully determined, and the accuracy of the STR genotyping was verified by the gold standard. Specifically, the typing success rate for 1150 genotypes was 95.3%, and the accuracy rate was 86.87%. Although this study focused primarily on demonstrating the feasibility of full-process testing, it can be optimistically predicted that further improvements in bioinformatics workflows and nanopore sequencing technology will help enhance the feasibility of Oxford Nanopore Technologies equipment for real-time individual identification at accident sites.

Carriage and within-host diversity of mcr-1.1-harbouring Escherichia coli from pregnant mothers: inter- and intra-mother transmission dynamics of mcr-1.1.

Exchange of antimicrobial resistance genes via mobile genetic elements occur in the gut which can be transferred from mother to neonate during birth. This study is the first to analyse transmissible colistin resistance gene, mcr, in pregnant mothers and neonates. Samples were collected from pregnant mothers (rectal) and septicaemic neonates (rectal and blood) and analysed for the presence of mcr, its transmissibility, genome diversity, and exchange of mcr between isolates within an individual and across different individuals (not necessarily mother-baby pairs). mcr-1.1 was detected in rectal samples of pregnant mothers (n = 10, 0.9%), but not in neonates. All mcr-positive mothers gave birth to healthy neonates from whom rectal specimen were not collected. Hence, the transmission of mcr between these mother-neonate pairs could not be studied. mcr-1.1 was noted only in Escherichia coli (phylogroup A & B1), and carried few resistance and virulence genes. Isolates belonged to diverse sequence types (n = 11) with two novel STs (ST12452, ST12455). mcr-1.1 was borne on conjugative IncHI2 bracketed between ISApl1 on Tn6630, and the plasmids exhibited similarities in sequences across the study isolates. Phylogenetic comparison showed that study isolates were related to mcr-positive isolates of animal origin from Southeast Asian countries. Spread of mcr-1.1 within this study occurred either via similar mcr-positive clones or similar mcr-bearing plasmids in mothers. Though this study could not build evidence for mother-baby transmission but the presence of such genes in the maternal specimen may enhance the chances of transmission to neonates.

Minigene Splicing Assays and Long-Read Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital Aniridia.

PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis.

Spatiotemporal variation in urban wastewater pollution impacts on river microbiomes and associated hazards in the Akaki catchment, Addis Ababa, Ethiopia.

In Addis Ababa and its environs, most urban wastewater is discharged into rivers without treatment. This study related urban wastewater characteristics to the prevalence of faecal, antibiotic resistant, and potentially pathogenic bacteria in rivers of the Akaki catchment across six locations, for the dry and wet season. Spatiotemporal variation in bacterial hazards across the catchment was up to 6 log10 units. Cooccurrence of sewage pollution marker gene HF183 in all river samples testing positive for the Vibrio cholerae marker gene ompW, and high levels of these two genes in untreated wastewater, identified human sewage as the likely source of Vibrio cholerae hazards in the catchment. Levels of the marker genes rodA for E. coli, HF183 for human host associated Bacteroides, ciaB for Arcobacter, and ompW for Vibrio cholerae were all higher in the dry season than in the wet season. Marker gene gyrB for Pseudomonas aeruginosa was not detected in the samples. From the sequencing data, notable bacterial genera in the dry season included wastewater pollution indicators Arcobacter and Aeromonas, whereas soil erosion may explain the greater prominence of Legionella, Vicinamibacter, and Sphingomonas during the wet season. Except for the most upstream location, all faecal coliform (FC) counts exceeded WHO standards of 1000 CFU/100 mL for unrestricted irrigation. Concerningly, 0.6-20% of FC had ESBL producing antimicrobial resistance traits. In conclusion, multiple bacterial hazards were of concern for river water users in the Akaki catchment, and elevated in the dry season, when the river water is being used for irrigation of vegetable fields that supply the markets of Addis Ababa. This reflects inadequate treatment and limited dilution of urban wastewater by the natural river flows during periods of low rainfall.

Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles.

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re-sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance-associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty-eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole-genome-sequencing and Sanger-sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance-associated mutations detected by MinION-nanopore-sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.

Comprehensive Pathogen Identification, Antibiotic Resistance, and Virulence Genes Prediction Directly From Simulated Blood Samples and Positive Blood Cultures by Nanopore Metagenomic Sequencing.

Bloodstream infection is a major cause of morbidity and mortality worldwide. We explored whether MinION nanopore sequencing could accelerate diagnosis, resistance, and virulence profiling prediction in simulated blood samples and blood cultures. One milliliter of healthy blood samples each from direct spike (sample 1), anaerobic (sample 2), and aerobic (sample 3) blood cultures with initial inoculation of ∼30 CFU/ml of a clinically isolated Klebsiella pneumoniae strain was subjected to DNA extraction and nanopore sequencing. Hybrid assembly of Illumina and nanopore reads from pure colonies of the isolate (sample 4) was used as a reference for comparison. Hybrid assembly of the reference genome identified a total of 39 antibiotic resistance genes and 77 virulence genes through alignment with the CARD and VFDB databases. Nanopore correctly detected K. pneumoniae in all three blood samples. The fastest identification was achieved within 8 h from specimen to result in sample 1 without blood culture. However, direct sequencing in sample 1 only identified seven resistance genes (20.6%) but 28 genes in samples 2-4 (82.4%) compared to the reference within 2 h of sequencing time. Similarly, 11 (14.3%) and 74 (96.1%) of the virulence genes were detected in samples 1 and 2-4 within 2 h of sequencing time, respectively. Direct nanopore sequencing from positive blood cultures allowed comprehensive pathogen identification, resistance, and virulence genes prediction within 2 h, which shows its promising use in point-of-care clinical settings.

Molecular Approaches for Low-Cost Point-of-Care Pathogen Detection in Agriculture and Forestry.

Early detection of plant diseases is a crucial factor to prevent or limit the spread of a rising infection that could cause significant economic loss. Detection test on plant diseases in the laboratory can be laborious, time consuming, expensive, and normally requires specific technical expertise. Moreover, in the developing countries, it is often difficult to find laboratories equipped for this kind of analysis. Therefore, in the past years, a high effort has been made for the development of fast, specific, sensitive, and cost-effective tests that can be successfully used in plant pathology directly in the field by low-specialized personnel using minimal equipment. Nucleic acid-based methods have proven to be a good choice for the development of detection tools in several fields, such as human/animal health, food safety, and water analysis, and their application in plant pathogen detection is becoming more and more common. In the present review, the more recent nucleic acid-based protocols for point-of-care (POC) plant pathogen detection and identification are described and analyzed. All these methods have a high potential for early detection of destructive diseases in agriculture and forestry, they should help make molecular detection for plant pathogens accessible to anyone, anywhere, and at any time. We do not suggest that on-site methods should replace lab testing completely, which remains crucial for more complex researches, such as identification and classification of new pathogens or the study of plant defense mechanisms. Instead, POC analysis can provide a useful, fast, and efficient preliminary on-site screening that is crucial in the struggle against plant pathogens.

Metagenomic water quality monitoring with a portable laboratory.

We describe the technical feasibility of metagenomic water quality analysis using only portable equipment, for example mini-vacuum pumps and filtration units, mini-centrifuges, mini-PCR machines and the memory-stick sized MinION of Oxford Nanopore Technologies, for the library preparation and sequencing of 16S rRNA gene amplicons. Using this portable toolbox on site, we successfully characterized the microbiome of water samples collected from Birtley Sewage Treatment Plant, UK, and its environs. We also demonstrated the applicability of the portable metagenomics toolbox in a low-income country by surveying water samples from the Akaki River around Addis Ababa, Ethiopia. The 16S rRNA gene sequencing workflow, including DNA extraction, PCR amplification, sequencing library preparation, and sequencing was accomplished within one working day. The metagenomic data became available within 24-72 h, depending on internet speed. Metagenomic analysis clearly distinguished the microbiome of pristine samples from sewage influenced water samples. Metagenomic analysis identified the potential role of two bacterial genera not conventionally monitored, Arcobacter and Aeromonas, as predominant faecal pollution indicators/waterborne hazards. Subsequent quantitative PCR analysis validated the high Arcobacter butzleri abundances observed in the urban influenced Akaki River water samples by portable next generation sequencing with the MinION device. Overall, our field deployable metagenomics toolbox advances the capability of scientists to comprehensively monitor microbiomes anywhere in the world, including in the water, food and drinks industries, the health services, agriculture and beyond.

Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform.

Real-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples' genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing.

Resolving plasmid structures in Enterobacteriaceae using the MinION nanopore sequencer: assessment of MinION and MinION/Illumina hybrid data assembly approaches.

This study aimed to assess the feasibility of using the Oxford Nanopore Technologies (ONT) MinION long-read sequencer in reconstructing fully closed plasmid sequences from eight Enterobacteriaceae isolates of six different species with plasmid populations of varying complexity. Species represented were Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia marcescens and Klebsiella oxytoca, with plasmid populations ranging from 1-11 plasmids with sizes of 2-330 kb. Isolates were sequenced using Illumina (short-read) and ONT's MinION (long-read) platforms, and compared with fully resolved PacBio (long-read) sequence assemblies for the same isolates. We compared the performance of different assembly approaches including SPAdes, plasmidSPAdes, hybridSPAdes, Canu, Canu+Pilon (canuPilon) and npScarf in recovering the plasmid structures of these isolates by comparing with the gold-standard PacBio reference sequences. Overall, canuPilon provided consistently good quality assemblies both in terms of assembly statistics (N50, number of contigs) and assembly accuracy [presence of single nucleotide polymorphisms (SNPs)/indels with respect to the reference sequence]. For plasmid reconstruction, Canu recovered 70 % of the plasmids in complete contigs, and combining three assembly approaches (Canu or canuPilon, hybridSPAdes and plasmidSPAdes) resulted in a total 78 % recovery rate for all the plasmids. The analysis demonstrated the potential of using MinION sequencing technology to resolve important plasmid structures in Enterobacteriaceae species independent of and in conjunction with Illumina sequencing data. A consensus assembly derived from several assembly approaches could present significant benefit in accurately resolving the greatest number of plasmid structures.

Application of highly portable MinION nanopore sequencing technology for the monitoring of nosocomial tuberculosis infection.

Referral hospitals in sub-Saharan Africa concentrate large numbers of tuberculosis (TB) and multidrug-resistant TB (MDR-TB) patients, failed by community TB services. We have previously shown, from enhanced screening and through autopsy studies, a significant burden of missed TB infections at the University Teaching Hospital, Lusaka, Zambia, with many patients dying or being discharged without treatment. With minimal TB isolation facilities and minimal political will to invest in broader screening and isolation, the risk of nosocomial transmission is likely to be extremely high. Studies from other hospitals in low burden settings and in South Africa have shown that next generation sequencing (NGS) is a very powerful tool for rapidly sequencing whole TB genomes and comparing them to confirm or rule out nosocomial transmission. The established platforms for NGS analysis, such as Illumina, are very expensive, immobile, and require regular maintenance, making them a costly inclusion on a research proposal or programmatic intervention grant in Africa. MinION nanopore sequencing has changed the NGS landscape with cheap portable sequencers, rapid simple library preparation (15min), and automated real-time analysis tools. The application of highly portable MinION nanopore sequencing technology for the monitoring of nosocomial TB infection will be discussed. Preliminary data from our pediatric pneumonia study will demonstrate the detection of TB in induced sputum from children admitted to the University Teaching Hospital.