MinION Sequencing Platform Research Articles

Evaluating the lettuce metatranscriptome with MinION sequencing for future spaceflight food production applications

Healthy plants are vital for successful, long-duration missions in space, as they provide the crew with life support, food production, and psychological benefits. The microorganisms that associate with plant tissues play a critical role in improving plant health and production. To that end, we developed a methodology to investigate the transcriptional activities of the microbiome of red romaine lettuce, a key salad crop that was grown under International Space Station (ISS)-like conditions. Microbial transcripts enriched from host–microbe total RNA were sequenced using the Oxford Nanopore MinION sequencing platform. Results show that this enrichment approach was highly reproducible and could be an effective approach for the on-site detection of microbial transcriptional activity. Our results demonstrate the feasibility of using metatranscriptomics of enriched microbial RNA as a potential method for on-site monitoring of the transcriptional activity of crop microbiomes, thereby helping to facilitate and maintain plant health for on-orbit space food production.

Phylogenomics reveals viral sources, transmission, and potential superinfection in early-stage COVID-19 patients in Ontario, Canada

The emergence and rapid global spread of SARS-CoV-2 demonstrates the importance of infectious disease surveillance, particularly during the early stages. Viral genomes can provide key insights into transmission chains and pathogenicity. Nasopharyngeal swabs were obtained from thirty-two of the first SARS-CoV-2 positive cases (March 18–30) in Kingston Ontario, Canada. Viral genomes were sequenced using Ion Torrent (n = 24) and MinION (n = 27) sequencing platforms. SARS-CoV-2 genomes carried forty-six polymorphic sites including two missense and three synonymous variants in the spike protein gene. The D614G point mutation was the predominate viral strain in our cohort (92.6%). A heterozygous variant (C9994A) was detected by both sequencing platforms but filtered by the ARTIC network bioinformatic pipeline suggesting that heterozygous variants may be underreported in the SARS-CoV-2 literature. Phylogenetic analysis with 87,738 genomes in the GISAID database identified global origins and transmission events including multiple, international introductions as well as community spread. Reported travel history validated viral introduction and transmission inferred by phylogenetic analysis. Molecular epidemiology and evolutionary phylogenetics may complement contact tracing and help reconstruct transmission chains of emerging diseases. Earlier detection and screening in this way could improve the effectiveness of regional public health interventions to limit future pandemics.

Characterization of a vancomycin-resistant Enterococcus faecium isolate and a vancomycin-susceptible E. faecium isolate from the same blood culture.

To characterize two Enterococcus faecium isolates with different resistance phenotypes obtained from the same blood culture. The isolates were identified by MALDI-TOF MS and antimicrobial susceptibility testing (AST) was performed using a VITEK® 2 AST P592 card and Etest. WGS was performed on the MiSeq and MinION sequencer platforms. Core-genome MLST (cgMLST) and seven-loci MLST were performed. Plasmid analysis was performed using S1-PFGE followed by Southern-blot hybridization. Both E. faecium isolates were ST203. AST revealed that one was a vancomycin-resistant E. faecium (VREfm) isolate and the other was a vancomycin-susceptible E. faecium (VSEfm) isolate. The VREfm isolate harboured the vanA gene cluster as part of a Tn1546-type transposon encoded on a 49 kb multireplicon (rep1, rep2 and rep7a) plasmid (pAML0157.1). On the same plasmid, ant(6)-Ia, cat-like and erm(B) were encoded. The VSEfm isolate harboured a rep2 plasmid (pAML0158.1), 12 kb in size, which was present in full length as part of pAML0157.1 from the VREfm isolate. The vanA-encoding pAML0157.1 was a chimera of the rep2 pAML0158.1 and a second DNA segment harbouring vanA, ant(6)-Ia, erm(B) and cat-like, as well as the replicons rep1 and rep7a. By cgMLST analysis, the VREfm and VSEfm isolates were identical. Our results demonstrate that the VREfm and VSEfm blood culture isolates represented ST203 and were identical. The investigated heterogeneous resistance phenotypes resulted from the acquisition or loss of plasmid segments in the enterococcal isolates. These data illustrate that mobile genetic elements may contribute to the spread of vancomycin resistance among enterococci and to the genotypic and phenotypic variation within clonal isolates.

MODL-22. DEVELOPING A REAL-TIME PERSONALIZED DRUG TESTING PLATFORM FOR PEDIATRIC CNS CANCERS

INTRODUCTIONThe relatively small size of biopsied CNS tumors has presented a historical challenge for real-time drug screens. Moreover, in vivo assessment of drug response does not often benefit patients with aggressive gliomas given the relatively long time (>8 months) of tumor engraftment in the classic mouse PDX models. Here, we aimed to develop an innovative real-time in vivo and in vitro drug screening platform capable of analyzing a minimal number (<1E6) of cells obtained at biopsy.METHODSExisting primary cells were used to test 6 different culture platforms. The top platform was selected and used to expand tumor cells obtained of DMG biopsy. Tumor cells were validated using the minION sequencing platform. Single and combination drug (n=7) screens were performed. Effective drugs were further evaluated in zebrafish PDX and non-tumor bearing models to assess efficacy and toxicity, respectively.RESULTSA total of 8 biopsies were obtained. Successful cell expansion was achieved in 6/8 (75%) and a limited drug screen in 3/6 (50%) of cases. Single and combination drug (n=7) assays identified responder and non-responders to candidate drugs. Systemic toxicity of effective drugs was tested in non-tumor bearing zebrafish. Tumor cells were engrafted in zebrafish providing the opportunity for an in vivo screen. The entire process was completed within 21 days on average.CONCLUSIONSA novel platform was developed for rapid in vitro and in vivo drug screens of tumor cells obtained at biopsy. This platform will provide the opportunity to establish personalized therapy for heterogeneous cancers including DMGs.

Chromosome-level de novo assembly of Coprinopsis cinerea A43mut B43mut pab1-1 #326 and genetic variant identification of mutants using Nanopore MinION sequencing

The homokaryotic Coprinopsis cinerea strain A43mut B43mut pab1-1 #326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factors A and B result in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with the C. cinerea strain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein (RasGAP) was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform.

A hybrid plasmid formed by recombination of a virulence plasmid and a resistance plasmid in Klebsiella pneumoniae

ObjectiveThe emergence of multidrug-resistant (MDR) and hypervirulent Klebsiella pneumoniae (hvKP) facilitates simultaneous dissemination of virulence and resistance in a single event, which poses serious threat to public health. MethodsThis study characterized the multidrug-resistant and moderately virulent ST11 K64 K. pneumoniae strain HB25-1 from a clinical case with microbiological and genomic approaches. Plasmids from strain HB25-1 were subjected to whole plasmid sequencing using both the Illumina NextSeq 500 sequencing platform and Nanopore MinION sequencer platforms. Klebsiella pneumoniae HB25-1 was subjected to a conjugation experiment and Galleria mellonella infection model to evaluate the transmission and virulence potential. ResultsWe report the emergence of an ST11, serotype K64 K. pneumoniae isolate, which is resistant to third-generation cephalosporin and exhibited a moderate level of virulence. WGS revealed that this strain harboured a plasmid, pHB25-1, which carried multidrug resistance genes (blaDHA-1, qnrB4, dfrA12, aadA2, sul1, aac(3)-lld, blaTEM-1, mph(E)) and virulence-encoding genes (the regulator of mucoid phenotype A gene rmpA2 and the aerobactin gene cluster iutAiucABCD). Genomic analysis indicated that pHB25-1 was formed through co-integration of structural regions located in two different plasmids, enabling it to encode both resistance and virulent phenotypes. ConclusionFindings in this study provide evidence of active plasmid evolution in K. pneumoniae and suggest that surveillance of multidrug-resistant and hypervirulent K. pneumoniae is urgently needed.

Characterization of Fecal Microbiota with Clinical Specimen Using Long-Read and Short-Read Sequencing Platform.

Accurate and rapid identification of microbiotic communities using 16S ribosomal (r)RNA sequencing is a critical task for expanding medical and clinical applications. Next-generation sequencing (NGS) is widely considered a practical approach for direct application to communities without the need for in vitro culturing. In this report, a comparative evaluation of short-read (Illumina) and long-read (Oxford Nanopore Technologies (ONT)) platforms toward 16S rRNA sequencing with the same batch of total genomic DNA extracted from fecal samples is presented. Different 16S gene regions were amplified, bar-coded, and sequenced using the Illumina MiSeq and ONT MinION sequencers and corresponding kits. Mapping of the sequenced amplicon using MinION to the entire 16S rRNA gene was analyzed with the cloud-based EPI2ME algorithm. V3–V4 reads generated using MiSeq were aligned by applying the CLC genomics workbench. More than 90% of sequenced reads generated using distinct sequencers were accurately classified at the genus or species level. The misclassification of sequenced reads at the species level between the two approaches was less substantial as expected. Taken together, the comparative results demonstrate that MinION sequencing platform coupled with the corresponding algorithm could function as a practicable strategy in classifying bacterial community to the species level.

First record of a tandem-repeat region within the mitochondrial genome of Clonorchis sinensis using a long-read sequencing approach.

BackgroundMitochondrial genomes provide useful genetic markers for systematic and population genetic studies of parasitic helminths. Although many such genome sequences have been published and deposited in public databases, there is evidence that some of them are incomplete relating to an inability of conventional techniques to reliably sequence non-coding (repetitive) regions. In the present study, we characterise the complete mitochondrial genome—including the long, non-coding region—of the carcinogenic Chinese liver fluke, Clonorchis sinensis, using long-read sequencing.MethodsThe mitochondrial genome was sequenced from total high molecular-weight genomic DNA isolated from a pool of 100 adult worms of C. sinensis using the MinION sequencing platform (Oxford Nanopore Technologies), and assembled and annotated using an informatic approach.ResultsFrom > 93,500 long-reads, we assembled a 18,304 bp-mitochondrial genome for C. sinensis. Within this genome we identified a novel non-coding region of 4,549 bp containing six tandem-repetitive units of 719–809 bp each. Given that genomic DNA from pooled worms was used for sequencing, some variability in length/sequence in this tandem-repetitive region was detectable, reflecting population variation.ConclusionsFor C. sinensis, we report the complete mitochondrial genome, which includes a long (> 4.5 kb) tandem-repetitive region. The discovery of this non-coding region using a nanopore-sequencing/informatic approach now paves the way to investigating the nature and extent of length/sequence variation in this region within and among individual worms, both within and among C. sinensis populations, and to exploring whether this region has a functional role in the regulation of replication and transcription, akin to the mitochondrial control region in mammals. Although applied to C. sinensis, the technological approach established here should be broadly applicable to characterise complex tandem-repetitive or homo-polymeric regions in the mitochondrial genomes of a wide range of taxa.

Improving the Chromosome-Level Genome Assembly of the Siamese Fighting Fish (Betta splendens) in a University Master's Course.

Ever decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish (Betta splendens) that was carried out during a practical university master’s course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behavior. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published Hi-C data. The use of ∼35x nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using the Hi-C data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 96.1% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly. We present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university master’s course. The use of ∼35× long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.

Complete, high-quality genomes from long-read metagenomic sequencing of two wolf lichen thalli reveals enigmatic genome architecture

Fungal genomes display incredible levels of complexity and diversity, and are exceptional study systems for genome evolution. Here we used the Oxford Nanopore MinION sequencing platform to generate high-quality fungal genomes from complex metagenomic samples of lichen thalli. We sequenced two wolf lichens using one flow cell per sample, generating 17.1 Gbps for Letharia lupina and 14.3 Gbps for Letharia columbiana. The resulting L. lupina genome is one of the most contiguous lichen genomes available to date, with 49.2 Mbp contained on 31 contigs. The L. columbiana genome, while less contiguous, is still relatively high quality, with 52.3 Mbp on a total of 161 contigs. Each thallus for both species contained multiple distinct haplotypes, a phenomenon that has rarely been empirically demonstrated. The Oxford Nanopore sequencing technologies are robust and effective when applied to complex symbioses, and have the potential to fundamentally transform our understanding of fungal genetics.

Whole genome sequencing and antibiotic diffusion assays, provide new insight on drug resistance in the genus Pedobacter.

ABSTRACTA total of four strains of the ‘environmental superbug’ Pedobacter isolated from sludge produced at Norwegian drinking water treatment plants, were characterized by whole genome sequencing and antibiotic susceptibility assays. As with previous studies on members of this genus, we found that the isolates were multi-drug resistant, and that this resistance included clinically important beta-lactams, aminoglycosides and the fluoroquinolone ciprofloxacin. Using the minION sequencing platform (Oxford Nanopore Technologies) combined with HiSeq PE150 Illumina sequencing data, the four isolates were assembled into genomes of single contigs. Analysis of the genomes revealed potential genetic factors possibly underlying some of the specific resistances observed. Metallo-beta-lactamase activity was detected in one isolate, and the same isolate contained a putative metallo-betalactamase gene resembling pedo-2. Furthermore, several genes related to multidrug efflux systems were found using the resistance database CARD. Additionally, the present study extends our knowledge on the phylogeny of this genus, adding four new genomes to the existing 50.

The Application of Nanopore Sequencing Technology to the Study of Dinoflagellates: A Proof of Concept Study for Rapid Sequence-Based Discrimination of Potentially Harmful Algae.

Harmful algal blooms (HABs) are a naturally occurring global phenomena that have the potential to impact fisheries, leisure and ecosystems, as well as posing a significant hazard to animal and human health. There is significant interest in the development and application of methodologies to study all aspects of the causative organisms and toxins associated with these events. This paper reports the first application of nanopore sequencing technology for the detection of eukaryotic harmful algal bloom organisms. The MinION sequencing platform from Oxford Nanopore technologies provides long read sequencing capabilities in a compact, low cost, and portable format. In this study we used the MinION to sequence long-range PCR amplicons from multiple dinoflagellate species with a focus on the genus Alexandrium. Primers applicable to a wide range of dinoflagellates were selected, meaning that although the study was primarily focused on Alexandrium the applicability to three additional genera of toxic algae, namely; Gonyaulax, Prorocentrum, and Lingulodinium was also demonstrated. The amplicon generated here spanned approximately 3 kb of the rDNA cassette, including most of the 18S, the complete ITS1, 5.8S, ITS2 and regions D1 and D2 of the 28S. The inclusion of barcode genes as well as highly conserved regions resulted in identification of organisms to the species level. The analysis of reference cultures resulted in over 99% of all sequences being attributed to the correct species with an average identity above 95% from a reference list of over 200 species (see Supplementary Material 1). The use of mock community analysis within environmental samples highlighted that complex matrices did not prevent the ability to distinguish between phylogenetically similar species. Successful identification of causative organisms in environmental samples during natural toxic events further highlighted the potential of the assay. This study proves the suitability of nanopore sequencing technology for taxonomic identification of harmful algal bloom organisms and acquisition of data relevant to the World Health Organisations “one health” approach to marine monitoring.

Early adaptation strategies of Saccharomyces cerevisiae and Torulaspora delbrueckii to co-inoculation in high sugar grape must-like media

Torulaspora delbrueckii and Saccharomyces cerevisiae are yeast species found concurrently in wine. In order to commence fermentation, they adapt to the initial harsh environment, maintaining cellular homeostasis and promoting metabolism. These actions involve an intricate regulation of stress tolerance, growth and metabolic genes. Their phenotypes are influenced by the fermentation environment and physiological state of the cell, but such gene-environment interactions are poorly understood. This study aimed to compare the cell physiology of the two species, through genome-wide analysis of gene expression, coupling Oxford Nanopore MinION and Illumina Hiseq sequencing platforms. The early transcriptional responses to stress, nutrients and cell-to-cell communication were analysed. Particular attention was given to the fundamental gene modulations, leading to an understanding of the physiological changes needed to maintain cellular homeostasis, exit the quiescent state and establish dominance in the fermentation. Our findings suggest the existence of species-specific adaptation strategies in response to growth in a high sugar synthetic grape juice medium.

Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens

The MinION sequencing platform offers near real-time analysis of DNA sequence; this makes the tool attractive for deployment in fieldwork or clinical settings. We used the MinION platform coupled to the NanoOK RT software package to perform shotgun metagenomic sequencing and profile mock communities and faecal samples from healthy and ill preterm infants. Using Nanopore data, we reliably classified a 20-species mock community and captured the diversity of the immature gut microbiota over time and in response to interventions such as probiotic supplementation, antibiotic treatment or episodes of suspected sepsis. We also performed rapid real-time runs to assess gut-associated microbial communities in critically ill and healthy infants, facilitated by NanoOK RT software package, which analysed sequences as they were generated. Our pipeline reliably identified pathogenic bacteria (that is, Klebsiella pneumoniae and Enterobacter cloacae) and their corresponding antimicrobial resistance gene profiles within as little as 1 h of sequencing. Results were confirmed using pathogen isolation, whole-genome sequencing and antibiotic susceptibility testing, as well as mock communities and clinical samples with known antimicrobial resistance genes. Our results demonstrate that MinION (including cost-effective Flongle flow cells) with NanoOK RT can process metagenomic samples to a rich dataset in < 5 h, which creates a platform for future studies aimed at developing these tools and approaches in clinical settings with a focus on providing tailored patient antimicrobial treatment options.

Shotgun metagenome data of a defined mock community using Oxford Nanopore, PacBio and Illumina technologies

Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2–7.2 Mbp, 40–73% GC content, and 1.5–7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina + ONT and Illumina + PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel.

Toward on-site food authentication using nanopore sequencing

•MinION DNA metabarcoding is a promising tool for species identification in food.•MinION and Illumina MiSeq sequencing platforms perform equally accurate.•Species identification with MinION sequencing requires dedicated bioinformatics.

Identification and characterization of a conjugative blaVIM-1-bearing plasmid in Vibrio alginolyticus of food origin.

To investigate the genetic features of the blaVIM-1 gene first detected in a cephalosporin-resistant Vibrio alginolyticus isolate, Vb1978. The MICs of V. alginolyticus strain Vb1978 were determined, and the β-lactamases produced were screened and analysed using conjugation, S1-PFGE and Southern blotting. The complete sequence of the blaVIM-1-encoding plasmid was also obtained using the Illumina and MinION sequencing platforms. V. alginolyticus strain Vb1978, isolated from a retail shrimp sample, was resistant to cephalosporins and exhibited reduced susceptibility to carbapenems. A novel blaVIM-1-carrying conjugative plasmid, designated pVb1978, was identified in this strain. Plasmid pVb1978 had 50 001 bp and comprised 59 predicted coding sequences (CDSs). The plasmid backbone of pVb1978 was homologous to those of IncP-type plasmids, while its replication region was structurally similar to non-IncP plasmids. The blaVIM-1 gene was found to be carried by the class 1 integron In70 and associated with a defective Tn402-like transposon. A novel blaVIM-1-carrying conjugative plasmid, pVb1978, was reported for the first time in V. alginolyticus, which warrants further investigation in view of its potential pathogenicity towards humans and widespread occurrence in the environment.

Long-Read Annotation: Automated Eukaryotic Genome Annotation Based on Long-Read cDNA Sequencing.

Single-molecule full-length complementary DNA (cDNA) sequencing can aid genome annotation by revealing transcript structure and alternative splice forms, yet current annotation pipelines do not incorporate such information. Here we present long-read annotation (LoReAn) software, an automated annotation pipeline utilizing short- and long-read cDNA sequencing, protein evidence, and ab initio prediction to generate accurate genome annotations. Based on annotations of two fungal genomes (Verticillium dahliae and Plicaturopsis crispa) and two plant genomes (Arabidopsis [Arabidopsis thaliana] and Oryza sativa), we show that LoReAn outperforms popular annotation pipelines by integrating single-molecule cDNA-sequencing data generated from either the Pacific Biosciences or MinION sequencing platforms, correctly predicting gene structure, and capturing genes missed by other annotation pipelines.

Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome

For the first time, a coding complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabeled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza A virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method with total RNA extracted from the allantoic fluid of influenza rA/Puerto Rico/8/1934 (H1N1) virus infected chicken eggs (EID50 6.8 × 109), we demonstrate successful sequencing of the coding complete influenza A virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza A virus. By utilizing the same methodology one can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle, or other pathogens. This approach also has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

Structural, Physiological and Regulatory Analysis of Maltose Transporter Genes in Saccharomyces eubayanus CBS 12357T.

Saccharomyces pastorianus lager brewing yeasts are domesticated hybrids of Saccharomyces cerevisiae and cold-tolerant Saccharomyces eubayanus. To understand the contribution of both parental genomes to maltose metabolism in brewing wort, this study focuses on maltose transport in the S. eubayanus type strain CBS 12357T/FM1318. To obtain complete sequences of the MAL loci of this strain, a near-complete genome assembly was generated using the Oxford Nanopore Technology MinION sequencing platform. Except for CHRXII, all sixteen chromosomes were assembled as single contigs. Four loci harboring putative maltose transporter genes (SeMALT1-4), located in subtelomeric regions of CHRII, CHRV, CHRXIII, and CHRXVI, were completely resolved. The near-identical loci on CHRV and CHRXVI strongly resembled canonical S. cerevisiae MAL loci, while those on CHRII and CHRXIII showed different structures suggestive of gene loss. Overexpression of SeMALT1-4 in a maltose-transport-deficient S. cerevisiae strain restored growth on maltose, but not on maltotriose, indicating maltose-specific transport functionality of all four transporters. Simultaneous CRISPR-Cas9-assisted deletion of only SeMALT2 and SeMALT4, which shared 99.7% sequence identity, eliminated growth of S. eubayanus CBS 12357T on maltose. Transcriptome analysis of S. eubayanus CBS 12357T established that SeMALT1 and SeMALT3, are poorly expressed in maltose-grown cultures, while SeMALT2 and SeMALT4 were expressed at much higher levels than SeMALT1 and SeMALT3, indicating that only SeMALT2/4 are responsible for maltose consumption in CBS 12357T. These results represent a first genomic and physiological characterization of maltose transport in S. eubayanus CBS 12357T and provides a valuable resource for further industrial exploitation of this yeast.