ONT MinION Research Articles

P-397. Rapid Prediction of Genetic Relatedness of Escherichia coli Direct from Critical Care Samples Using Metagenomic Sequencing Paired with Neighbour Typing

Abstract Background Determining relatedness of bacterial pathogens within hospitals is important to rule out transmission events, but current approaches are typically slow and/or resource intensive. We sought to assess the utility of rapid long-read sequencing with nearest neighbour-typing for identifying relatedness direct from specimens. Figure 1. Plot of true and predicted genetic distances for E. coli. (a) All comparisons. (B) Only concordant comparisons. (C) All comparisons with LS<0.5. (D) All comparisons with LS>0.5. Green data points are predictions based on concordant calls, and orange data points are predictions based on discordant calls. Methods We sequenced remnant urine (n=71) and respiratory (n=7) samples with confirmed Escherichia coli (n=78) using ONT MinION, from critical care units at two large tertiary care institutions in Ontario, Canada. The resistance RASE method was used to predict the nearest neighbour for all samples. Using the nearest neighbours and the known reference database phylogeny, we predicted genetic (pairwise) distances (percentage). We then compared the predicted genetic distances with the true genetic distances. Plots visualizing the true and predicted genetic distances were generated and linear models performed. Genetic distance predictions were categorized as: (1) concordant (matching predicted and true sequence types) vs. discordant; and (2) high confidence in prediction (lineage score [LS] >0.5) vs. low confidence in prediction (LS < 0.5). Results For all specimens, predicted genetic distances demonstrated a modest but significant correlation (R2=0.52, p< 0.001) with true genetic distances (Fig 1A). Predictions of genetic distance were most accurate when the predicted sequence type matched the true sequence type (R2=0.99, p< 0.001) (Fig 1B). Using the lineage score improved the accuracy of genetic distance predictions (R2=0.744, p< 0.001) (Fig 1C-D). Using a genetic distance cut-off of 0.002 to denote unrelated (non-transmitted) events, as determined by the frequency distribution of genetic distances, a lineage score informed approach could identify genetically unrelated E. coli with a specificity of 0.99, sensitivity of 0.96, and positive predictive value of 0.99. Conclusion Rapid predictions of genetic-relatedness can be made directly from specimens using long-read sequencing paired with neighbour-typing. We demonstrate that in high confidence calls, the potential for related transmission between infections can be effectively ruled out. Disclosures Allison McGeer, MD, AstraZeneca: Honoraria|GSK: Honoraria|Merck: Honoraria|Moderna: Honoraria|Novavax: Honoraria|Pfizer: Grant/Research Support|Pfizer: Honoraria|Roche: Honoraria|Seqirus: Grant/Research Support|Seqirus: Honoraria

Capturing clinically relevant Campylobacter attributes through direct whole genome sequencing of stool.

Campylobacter is the leading bacterial cause of infectious intestinal disease, but the pathogen typically accounts for a very small proportion of the overall stool microbiome in each patient. Diagnosis is even more difficult due to the fastidious nature of Campylobacter in the laboratory setting. This has, in part, driven a change in recent years, from culture-based to rapid PCR-based diagnostic assays which have improved diagnostic detection, whilst creating a knowledge gap in our clinical and epidemiological understanding of Campylobacter genotypes - no isolates to sequence. In this study, direct metagenomic sequencing approaches were used to assess the possibility of replacing genome sequences with metagenome sequences; metagenomic sequencing outputs were used to describe clinically relevant attributes of Campylobacter genotypes. A total of 37 diarrhoeal stool samples with Campylobacter and five samples with an unknown pathogen result were collected and processed with and without filtration, DNA was extracted, and metagenomes were sequenced by short-read sequencing. Culture-based methods were used to validate Campylobacter metagenome-derived genome (MDG) results. Sequence output metrics were assessed for Campylobacter genome quality and accuracy of characterization. Of the 42 samples passing quality checks for analysis, identification of Campylobacter to the genus and species level was dependent on Campylobacter genome read count, coverage and genome completeness. A total of 65% (24/37) of samples were reliably identified to the genus level through Campylobacter MDG, 73% (27/37) by culture and 97% (36/37) by qPCR. The Campylobacter genomes with a genome completeness of over 60% (n=21) were all accurately identified at the species level (100%). Of those, 72% (15/21) were identified to sequence types (STs), and 95% (20/21) accurately identified antimicrobial resistance (AMR) gene determinants. Filtration of stool samples enhanced Campylobacter MDG recovery and genome quality metrics compared to the corresponding unfiltered samples, which improved the identification of STs and AMR profiles. The phylogenetic analysis in this study demonstrated the clustering of the metagenome-derived with culture-derived genomes and revealed the reliability of genomes from direct stool sequencing. Furthermore, Campylobacter genome spiking percentages ranging from 0 to 2% total metagenome abundance in the ONT MinION sequencer, configured to adaptive sequencing, exhibited better assembly quality and accurate identification of STs, particularly in the analysis of metagenomes containing 2 and 1% of Campylobacter jejuni genomes. Direct sequencing of Campylobacter from stool samples provides clinically relevant and epidemiologically important genomic information without the reliance on cultured genomes.

Facilitating taxonomy and phylogenetics: An informative and cost-effective protocol integrating long amplicon PCRs and third-generation sequencing

Phylogenetic inference has become a standard technique in integrative taxonomy and systematics, as well as in biogeography and ecology. DNA barcodes are often used for phylogenetic inference, despite being strongly limited due to their low number of informative sites. Also, because current DNA barcodes are based on a fraction of a single, fast-evolving gene, they are highly unsuitable for resolving deeper phylogenetic relationships due to saturation. In recent years, methods that analyse hundreds and thousands of loci at once have improved the resolution of the Tree of Life, but these methods require resources, experience and molecular laboratories that most taxonomists do not have. This paper introduces a PCR-based protocol that produces long amplicons of both slow- and fast-evolving unlinked mitochondrial and nuclear gene regions, which can be sequenced by the affordable and portable ONT MinION platform with low infrastructure or funding requirements. As a proof of concept, we inferred a phylogeny of a sample of 63 spider species from 20 families using our proposed protocol. The results were overall consistent with the results from approaches based on hundreds and thousands of loci, while requiring just a fraction of the cost and labour of such approaches, making our protocol accessible to taxonomists worldwide.

Genomic DNA extraction optimization and validation for genome sequencing using the marine gastropod Kellet’s whelk

Next-generation sequencing technologies, such as Nanopore MinION, Illumina Hiseq and Novaseq, and PacBio Sequel II, hold immense potential for advancing genomic research on non-model organisms, including the vast majority of marine species. However, application of these technologies to marine invertebrate species is often impeded by challenges in extracting and purifying their genomic DNA due to high polysaccharide content and other secondary metabolites. In this study, we help resolve this issue by developing and testing DNA extraction protocols for Kellet’s whelk (Kelletia kelletii), a subtidal gastropod with ecological and commercial importance, by comparing four DNA extraction methods commonly used in marine invertebrate studies. In our comparison of extraction methods, the Salting Out protocol was the least expensive, produced the highest DNA yields, produced consistent high DNA quality, and had low toxicity. We validated the protocol using an independent set of tissue samples, then applied it to extract high-molecular-weight (HMW) DNA from over three thousand Kellet’s whelk tissue samples. The protocol demonstrated scalability and, with added clean-up, suitability for RAD-seq, GT-seq, as well as whole genome sequencing using both long read (ONT MinION) and short read (Illumina NovaSeq) sequencing platforms. Our findings offer a robust and versatile DNA extraction and clean-up protocol for supporting genomic research on non-model marine organisms, to help mediate the under-representation of invertebrates in genomic studies.

Whole genome sequencing of Vibrio harveyi from different sites in the Mediterranean Sea providing data on virulence and antimicrobial resistance genes

Vibriosis caused by Vibrio harveyi is an important bacterial infection in the Mediterranean farming of European seabass and gilthead sea bream. Genomic information about V. harveyi contributes to understanding its pathogenesis and virulence. Sixteen V. harveyi strains obtained from diseased seabass and sea bream from Croatia, France, Italy, Spain, Tunisia, and Turkey were sequenced using ONT MinION and Illumina HiSeq. The strains were sequenced, de novo assembled, and the genomes analysed for the presence of plasmids, virulence, and antibiotic resistance genes. Antibiotic resistance genes were compared with in vitro susceptibility testing using disc diffusion and microdilution methods. All studied strains had two circular chromosomes, and twelve strains had one to five circular plasmids. A total of 171 virulence genes were determined using the Virulence Factors of Bacterial Pathogens database (VFBD), 150 virulence genes were present on average in each genome and the distribution of virulence genes was similar across strains. Most of the reported virulence genes have been previously described in bacteria of the genus Vibrio, while genes sitA (4/16), sitB (3/16), sitC (4/16), and sitD (3/16) important for iron transport, and the ast gene (10/16) that encodes a heat-stable cytotoxic enterotoxin, were unique in our studied strains. Using the Comprehensive Antibiotic Resistance database (CARD), it was noted that all strains carried the CRP gene encoding resistance to macrolides, fluoroquinolone, and penams, but showed sensitivity to enrofloxacin and florfenicol using in vitro tests. The adeF gene coding for resistance to fluoroquinolones and tetracyclines was detected in lower percentage in thirteen strains was supported with detected sensitivity to florfenicol and oxytetracycline using in vitro tests. The E.coli parE gene was detected in eleven strains that showed sensitivity to florfenicol and oxytetracycline. The gene APH(3”)Ib responsible for resistance to aminoglycosides was present in one strain, though all strains showed susceptibility to gentamicin by in vitro testing. The Tet(D) gene coding for resistance to tetracyclines was found in a single strain showing reduced sensitivity to oxytetracycline. All tested strains were resistant to ampicillin by in vitro testing in line with previously described innate resistance. Phylogenetic analysis based on whole genome sequences showed a clear separation and clustering of the tested strains from the Mediterranean region. The new genomic data and knowledge provided in this study increase the understanding of the pathogenicity and antimicrobial resistance patterns of V. harveyi strains in the Mediterranean region, enabling a common regional approach to prevention and mitigation of the disease.

Molecular characterization and phylogenetic analysis of highly pathogenic H5N1 clade 2.3.4.4b virus in Bosnia and Herzegovina

Influenza A virus continues to represent a growing problem affecting mainly birds but with an increasing number of mammal transmission events reported each year. Nevertheless, molecular characterization and phylogenetic analysis of influenza A viruses originating from all confirmed cases have not been systematically performed in all parts of the world. In this study, we investigated a sample originating from a mute swan that died in November 2021 in the northern part of Bosnia and Herzegovina with RT-qPCR and whole genome sequencing using ONT MinION. It was diagnosed as a highly pathogenic Influenza A virus, subtype H5N1 of 2.3.4.4b clade, and phylogenetic analysis revealed high sequence homology with other European HPAI H5N1 sequences at the time. The notable detected mutations in HA (N110S and T139P) and NA genes (H155Y), that facilitate the host specificity shift and enable the resistance to some antiviral drugs respectively, underscore the necessity of virus evolution surveillance. Therefore, the rapid dissemination of information, including virological and molecular data, is essential for the introduction of tailored prevention measures for infected animals, providing clearer insight and better awareness of a potential public health threat.

Using custom-built primers and nanopore sequencing to evaluate CO-utilizer bacterial and archaeal populations linked to bioH2 production

The microbial community composition of five distinct thermophilic hot springs was effectively described in this work, using broad-coverage nanopore sequencing (ONT MinION sequencer). By examining environmental samples from the same source, but from locations with different temperatures, bioinformatic analysis revealed dramatic changes in microbial diversity and archaeal abundance. More specifically, no archaeal presence was reported with universal bacterial primers, whereas a significant archaea presence and also a wider variety of bacterial species were reported. These results revealed the significance of primer preference for microbiomes in extreme environments. Bioinformatic analysis was performed by aligning the reads to 16S microbial databases for identification using three different alignment methods, Epi2Me (Fastq 16S workflow), Kraken, and an in-house BLAST tool, including comparison at the genus and species levels. As a result, this approach to data analysis had a significant impact on the genera identified, and thus, it is recommended that use of multiple analysis tools to support findings on taxonomic identification using the 16S region until more precise bioinformatics tools become available. This study presents the first compilation of the ONT-based inventory of the hydrogen producers in the designated hot springs in Türkiye.

DNA barcoding, an effective tool for species identification: a review.

DNA barcoding is a powerful taxonomic tool to identify and discover species. DNA barcoding utilizes one or more standardized short DNA regions for taxon identification. With the emergence of new sequencing techniques, such as Next-generation sequencing (NGS), ONT MinION nanopore sequencing, and Pac Bio sequencing, DNA barcoding has become more accurate, fast, and reliable. Rapid species identification by DNA barcodes has been used in a variety of fields, including forensic science, control of the food supply chain, and disease understanding. The Consortium for Barcode of Life (CBOL) presents various working groups to identify the universal barcode gene, such as COI in metazoans; rbcL, matK, and ITS in plants; ITS in fungi; 16S rRNA gene in bacteria and archaea, and creating a reference DNA barcode library. In this article, an attempt has been made to analyze the various proposed DNA barcode for different organisms, strengths & limitations, recent advancements in DNA barcoding, and methods to speed up the DNA barcode reference library construction. This study concludes that constructing a reference library with high species coverage would be a major step toward identifying species by DNA barcodes. This can be achieved in a short period of time by using advanced sequencing and data analysis methods.

Occurrence of SARS-CoV-2 reinfections at regular intervals in Ecuador.

SARS-CoV-2 reinfection is defined as a new infection with a different virus variant in an individual who has already recovered from a previous episode of COVID-19. The first case of reinfection in the world was described in August 2020, since then, reinfections have increased over time and their incidence has fluctuated with specific SARS-CoV-2 variant waves. Initially, reinfections were estimated to represent less than 1% of total COVID-19 infections. With the advent of the Omicron variant, reinfections became more frequent, representing up to 10% of cases (based on data from developed countries). The frequency of reinfections in Latin America has been scarcely reported. The current study shows that in Ecuador, the frequency of reinfections has increased 10-fold following the introduction of Omicron, after 22 months of surveillance in a single center of COVID-19 diagnostics. Suspected reinfections were identified retrospectively from a database of RT-qPCR-positive patients. Cases were confirmed by sequencing viral genomes from the first and second infections using the ONT MinION platform. Monthly surveillance showed that the main incidence peaks of reinfections were reached within four to five months, coinciding with the increase of COVID-19 cases in the country, suggesting that the emergence of reinfections is related to higher exposure to the virus during outbreaks. This study performed the longest monitoring of SARS-CoV-2 reinfections, showing an occurrence at regular intervals of 4-5 months and confirming a greater propensity of Omicron to cause reinfections.

Evaluation of high molecular weight DNA extraction methods for long-read sequencing of Shiga toxin-producing Escherichia coli.

Next generation sequencing has become essential for pathogen characterization and typing. The most popular second generation sequencing technique produces data of high quality with very low error rates and high depths. One major drawback of this technique is the short reads. Indeed, short-read sequencing data of Shiga toxin-producing Escherichia coli (STEC) are difficult to assemble because of the presence of numerous mobile genetic elements (MGEs), which contain repeated elements. The resulting draft assemblies are often highly fragmented, which results in a loss of information, especially concerning MGEs or large structural variations. The use of long-read sequencing can circumvent these problems and produce complete or nearly complete genomes. The ONT MinION, for its small size and minimal investment requirements, is particularly popular. The ultra-long reads generated with the MinION can easily span prophages and repeat regions. In order to take full advantage of this technology it requires High Molecular Weight (HMW) DNA of high quality in high quantity. In this study, we have tested three different extraction methods: bead-based, solid-phase and salting-out, and evaluated their impact on STEC DNA yield, quality and integrity as well as performance in MinION long-read sequencing. Both the bead-based and salting-out methods allowed the recovery of large quantities of HMW STEC DNA suitable for MinION library preparation. The DNA extracted using the salting-out method consistently produced longer reads in the subsequent MinION runs, compared with the bead-based methods. While both methods performed similarly in subsequent STEC genome assembly, DNA extraction based on salting-out appeared to be the overall best method to produce high quantity of pure HMW STEC DNA for MinION sequencing.

First case of within-host co-infection of different SARS-CoV-2 variants in Ecuador

BackgroundCOVID-19 infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause mild symptoms to severe illness and death. Co-infections of SARS-CoV-2 with other respiratory viruses have been described. However, two SARS-CoV-2 lineage co-infection have been rarely reported. MethodologyA genotyping analysis and two different types of whole genome sequencing were performed (Illumina MiniSeq and ONT MinION). When examining the phylogenetic analysis in NextClade and Pangolin webservers, and considering the genotyping findings, conflicting results were obtained. ResultsThe raw data of the sequencing was analyzed, and nucleotide variants were identified between different reads of the virus genome. B.1 and P.1 lineages were identified within the same sample. ConclusionsWe concluded that this is a co-infection case with two SARS-CoV-2 lineages, the first one reported in Ecuador.

De Novo Assembly of the Dirofilaria immitis Genome by Long-Read Nanopore-Based Sequencing Technology on an Adult Worm from a Canine Cardiopulmonary Dirofilariosis Case.

Simple SummaryDirofilaria immitis is a zoonotic parasite that infects canids and other vertebrates. We expanded the use of long-read nanopore-based sequencing technology by performing genomic de novo assembly of a D. immitis specimen retrieved from a canine cardiopulmonary dirofilariasis case by using the ONT MinION platform. We also identified loci previously characterized as being associated to macrocyclic lactone resistance selection pressure. The identification of a resistant zoonotic parasite alerts for the overuse of macrocyclic lactone in the region.Dirofilaria immitis is a zoonotic parasitic nematode that infects domestic and wild canids, among its vertebrate hosts. The genetic analysis of D. immitis nowadays transcends the need for genetic taxonomy of nematodes, such as the study of resistance to macrocyclic lactone. We expanded the use of long-read nanopore-based sequencing technology on nematodes by performing genomic de novo assembly of a D. immitis specimen retrieved from a canine cardiopulmonary dirofilariasis case using the ONT MinION platform, followed by the study of macrocyclic lactone resistance. The assembled genome of D. immitis consists of 110 contigs with an N50 of 3687191. The genome size is 87899012 and contains a total of 9741 proteins; 6 ribosomal RNAs, with three belonging to the small subunit (18S) and three to the large subunit (28S); and 73 tRNAs. Subsequent analysis of six loci previously characterized as being associated to macrocyclic lactone resistance selection pressure showed that four have a genotype associated with either some loss of efficacy or the resistance phenotype. Considering the zoonotic potential of D. immitis, the identification of a resistant parasite alerts for the overuse of macrocyclic lactone in the region, which poses a potential risk to both veterinary and human public health.

Robust and scalable barcoding for massively parallel long-read sequencing

Nucleic-acid barcoding is an enabling technique for many applications, but its use remains limited in emerging long-read sequencing technologies with intrinsically low raw accuracy. Here, we apply so-called NS-watermark barcodes, whose error correction capability was previously validated in silico, in a proof of concept where we synthesize 3840 NS-watermark barcodes and use them to asymmetrically tag and simultaneously sequence amplicons from two evolutionarily distant species (namely Bordetella pertussis and Drosophila mojavensis) on the ONT MinION platform. To our knowledge, this is the largest number of distinct, non-random tags ever sequenced in parallel and the first report of microarray-based synthesis as a source for large oligonucleotide pools for barcoding. We recovered the identity of more than 86% of the barcodes, with a crosstalk rate of 0.17% (i.e., one misassignment every 584 reads). This falls in the range of the index hopping rate of established, high-accuracy Illumina sequencing, despite the increased number of tags and the relatively low accuracy of both microarray-based synthesis and long-read sequencing. The robustness of NS-watermark barcodes, together with their scalable design and compatibility with low-cost massive synthesis, makes them promising for present and future sequencing applications requiring massive labeling, such as long-read single-cell RNA-Seq.

3GOLD: optimized Levenshtein distance for clustering third-generation sequencing data

BackgroundThird-generation sequencing offers some advantages over next-generation sequencing predecessors, but with the caveat of harboring a much higher error rate. Clustering-related sequences is an essential task in modern biology. To accurately cluster sequences rich in errors, error type and frequency need to be accounted for. Levenshtein distance is a well-established mathematical algorithm for measuring the edit distance between words and can specifically weight insertions, deletions and substitutions. However, there are drawbacks to using Levenshtein distance in a biological context and hence has rarely been used for this purpose. We present novel modifications to the Levenshtein distance algorithm to optimize it for clustering error-rich biological sequencing data.ResultsWe successfully introduced a bidirectional frameshift allowance with end-user determined accommodation caps combined with weighted error discrimination. Furthermore, our modifications dramatically improved the computational speed of Levenstein distance. For simulated ONT MinION and PacBio Sequel datasets, the average clustering sensitivity for 3GOLD was 41.45% (S.D. 10.39) higher than Sequence-Levenstein distance, 52.14% (S.D. 9.43) higher than Levenshtein distance, 55.93% (S.D. 8.67) higher than Starcode, 42.68% (S.D. 8.09) higher than CD-HIT-EST and 61.49% (S.D. 7.81) higher than DNACLUST. For biological ONT MinION data, 3GOLD clustering sensitivity was 27.99% higher than Sequence-Levenstein distance, 52.76% higher than Levenshtein distance, 56.39% higher than Starcode, 48% higher than CD-HIT-EST and 70.4% higher than DNACLUST.ConclusionOur modifications to Levenshtein distance have improved its speed and accuracy compared to the classic Levenshtein distance, Sequence-Levenshtein distance and other commonly used clustering approaches on simulated and biological third-generation sequenced datasets. Our clustering approach is appropriate for datasets of unknown cluster centroids, such as those generated with unique molecular identifiers as well as known centroids such as barcoded datasets. A strength of our approach is high accuracy in resolving small clusters and mitigating the number of singletons.

Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing.

Rapid bacterial identification and antimicrobial resistance gene (ARG) detection are crucial for fast optimization of antibiotic treatment, especially for septic patients where each hour of delayed antibiotic prescription might have lethal consequences. This work investigates whether the Oxford Nanopore Technology’s (ONT) Flongle sequencing platform is suitable for real-time sequencing directly from blood cultures to identify bacteria and detect resistance-encoding genes. For the analysis, we used pure bacterial cultures of four clinical isolates of Escherichia coli and Klebsiella pneumoniae and two blood samples spiked with either E. coli or K. pneumoniae that had been cultured overnight. We sequenced both the whole genome and plasmids isolated from these bacteria using two different sequencing kits. Generally, Flongle data allow rapid bacterial ID and resistome detection based on the first 1,000–3,000 generated sequences (10 min to 3 h from the sequencing start), albeit ARG variant identification did not always correspond to ONT MinION and Illumina sequencing-based data. Flongle data are sufficient for 99.9% genome coverage within at most 20,000 (clinical isolates) or 50,000 (positive blood cultures) sequences generated. The SQK-LSK110 Ligation kit resulted in higher genome coverage and more accurate bacterial identification than the SQK-RBK004 Rapid Barcode kit.

Recovery of complete genomes and non-chromosomal replicons from activated sludge enrichment microbial communities with long read metagenome sequencing

New long read sequencing technologies offer huge potential for effective recovery of complete, closed genomes from complex microbial communities. Using long read data (ONT MinION) obtained from an ensemble of activated sludge enrichment bioreactors we recover 22 closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses, specifically microbes known to exhibit the polyphosphate- and glycogen-accumulating organism phenotypes (namely Candidatus Accumulibacter and Dechloromonas, and Micropruina, Defluviicoccus and Candidatus Contendobacter, respectively), and filamentous bacteria (Thiothrix) associated with the formation and stability of activated sludge flocs. Additionally we demonstrate the recovery of close to 100 circularised plasmids, phages and small microbial genomes from these microbial communities using long read assembled sequence. We describe methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes, and assess the influence of different correction procedures on genome quality and predicted gene quality. Our findings establish the feasibility of performing long read metagenome-assembled genome recovery for both chromosomal and non-chromosomal replicons, and demonstrate the value of parallel sampling of moderately complex enrichment communities to obtaining high quality reference genomes of key functional species relevant for wastewater bioprocesses.

Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes

Identification of human leukocyte antigen (HLA) alleles from next-generation sequencing (NGS) data is challenging because of the high polymorphism and mosaic nature of HLA genes. Owing to the complex nature of HLA genes and consequent challenges in allele assignment, Oxford Nanopore Technologies’ (ONT) single-molecule sequencing technology has been of great interest due to its fitness for sequencing long reads. In addition to the read length, ONT’s advantages are its portability and possibility for a rapid real-time sequencing, which enables a simultaneous data analysis. Here, we describe a targeted RNA-based method for HLA typing using ONT sequencing and SeqNext-HLA SeqPilot software (JSI Medical Systems GmbH). Twelve classical HLA genes were enriched from cDNA of 50 individuals, barcoded, pooled, and sequenced in 10 MinION R9.4 SpotON flow cell runs producing over 30,000 reads per sample. Using barcoded 2D reads, SeqPilot assigned HLA alleles to two-field typing resolution or higher with the average read depth of 1750x. Sequence analysis resulted in 99–100% accuracy at low-resolution level (one-field) and in 74–100% accuracy at high-resolution level (two-field) with the expected alleles. There are still some limitations with ONT RNA sequencing, such as noisy reads, homopolymer errors, and the lack of robust algorithms, which interfere with confident allele assignment. These issues need to be inspected carefully in the future to improve the allele call rates. Nevertheless, here we show that sequencing of multiplexed cDNA amplicon libraries on ONT MinION can produce accurate high-resolution typing results of 12 classical HLA loci. For HLA research, ONT RNA sequencing is a promising method due to its capability to sequence full-length HLA transcripts. In addition to HLA genotyping, the technique could also be applied for simultaneous expression analysis.

Human CHR18: “Stakhanovite” Genes, Missing and uPE1 Proteins in Liver Tissue and HepG2 Cells

Missing (MP) and functionally uncharacterized proteins (uPE1) comprise less than 5% of the total number of proteins encoded by human Chr18 genes. Within half a year, since the January 2020 version of NextProt, the number of entries in the MP+uPE1 datasets changed, mainly due to the achievements of antibody-based proteomics. Assuming that the proteome is closely related to the transcriptome scaffold, quantitative PCR, Illumina HiSeq, and Oxford Nanopore Technology were applied to characterize the liver samples of three male donors in comparison with the HepG2 cell line. The data mining of the Expression Atlas (EMBL-EBI) and the profiling of biopsy samples by using orthogonal methods of transcriptome analysis have shown that in HepG2 cells and the liver, the genes encoding functionally uncharacterized proteins (uPE1) are expressed as low as for the missing proteins (less than 1 copy per cell), except the selected cases of HSBP1L1, TMEM241, C18orf21, and KLHL14. The initial expectation that uPE1 genes might be expressed at higher levels than MP genes, was compromised by severe discrepancies in our semi-quantitative gene expression data and in public databanks. Such discrepancy forced us to revisit the transcriptome of Chr18, the target of the Russian C-HPP Consortium. Tanglegram of highly expressed genes and further correlation analysis have shown the severe dependencies on the mRNA extraction method and the analytical platform. Targeted gene expression analysis by quantitative PCR (qPCR) and high-throughput transcriptome profiling (Illumina HiSeq and ONT MinION) for the same set of samples from normal liver tissue and HepG2 cells revealed the detectable expression of 250+ (92%) protein-coding genes of Chr18 (at least one method). The expression of slightly more than 50% protein-coding genes was detected simultaneously by all three methods. Correlation analysis of the gene expression profiles showed that the grouping of the datasets depended almost equally on both the type of biological material and the experimental method, particularly cDNA/mRNA isolation and library preparation.

Antimicrobial Resistance and Genomic Characterization of OXA-48- and CTX-M-15-Co-Producing Hypervirulent Klebsiella pneumoniae ST23 Recovered from Nosocomial Outbreak.

Multidrug resistance (MDR) and hypervirulence (hv) have been long considered distinct evolutionary traits for Klebsiella pneumoniae (Kp), a versatile human pathogen. The recent emergence of Kp strains combining these traits poses a serious global threat. In this article, we describe the phenotypic and genomic characteristics of an MDR hvKp isolate, MAR14-456, representative of a nosocomial outbreak in Moscow, Russia, that was recovered from a postoperative wound in a patient who later developed multiple abscesses, fatal sepsis, and septic shock. Broth microdilution testing revealed decreased susceptibility of MAR14-456 to carbapenems (MICs 0.5–2 mg/L) and a high-level resistance to most β-lactams, β-lactam-β-lactamase-inhibitor combinations, and non-β-lactam antibiotics, except ceftazidime-avibactam, amikacin, tigecycline, and colistin. Whole-genome sequencing using Illumina MiSeq and ONT MinION systems allowed to identify and completely assemble two conjugative resistance plasmids, a typical ‘European’ epidemic IncL/M plasmid that carries the gene of OXA-48 carbapenemase, and an IncFIIK plasmid that carries the gene of CTX-M-15 ESBL and other resistance genes. MLST profile, capsular, lipopolysaccharide, virulence genes encoded on chromosome and IncHI1B/FIB plasmid, and the presence of apparently functional type I-E* CRISPR-Cas system were all characteristic of hvKp ST23, serotype K1-O1v2. Phylogenetic analysis showed the closest relatedness of MAR14-456 to ST23 isolates from China. This report highlights the threat of multiple resistance acquisition by hvKp strain and its spread as a nosocomial pathogen.

A Genomic Perspective on the Potential of Wild-Type Rumen Bacterium Enterobacter sp. LU1 as an Industrial Platform for Bio-Based Succinate Production.

Enterobacter sp. LU1, a wild-type bacterium originating from goat rumen, proved to be a potential succinic acid producer in previous studies. Here, the first complete genome of this strain was obtained and analyzed from a biotechnological perspective. A hybrid sequencing approach combining short (Illumina MiSeq) and long (ONT MinION) reads allowed us to obtain a single continuous chromosome 4,636,526 bp in size, with an average 55.6% GC content that lacked plasmids. A total of 4425 genes, including 4283 protein-coding genes, 25 ribosomal RNA (rRNA)-, 84 transfer RNA (tRNA)-, and 5 non-coding RNA (ncRNA)-encoding genes and 49 pseudogenes, were predicted. It has been shown that genes involved in transport and metabolism of carbohydrates and amino acids and the transcription process constitute the major group of genes, according to the Clusters of Orthologous Groups of proteins (COGs) database. The genetic ability of the LU1 strain to metabolize a wide range of industrially relevant carbon sources has been confirmed. The genome exploration indicated that Enterobacter sp. LU1 possesses all genes that encode the enzymes involved in the glycerol metabolism pathway. It has also been shown that succinate can be produced as an end product of fermentation via the reductive branch of the tricarboxylic acid cycle (TCA) and the glyoxylate pathway. The transport system involved in succinate excretion into the growth medium and the genes involved in the response to osmotic and oxidative stress have also been recognized. Furthermore, three intact prophage regions ~70.3 kb, ~20.9 kb, and ~49.8 kb in length, 45 genomic islands (GIs), and two clustered regularly interspaced short palindromic repeats (CRISPR) were recognized in the genome. Sequencing and genome analysis of Enterobacter sp. LU1 confirms many earlier results based on physiological experiments and provides insight into their genetic background. All of these findings illustrate that the LU1 strain has great potential to be an efficient platform for bio-based succinate production.