Single-molecule Sequencing Platform Research Articles

A Two-color Single-molecule Sequencing Platform and Its Clinical Applications.

DNA sequencers have become increasingly important research and diagnostic tools over the past 20 years. In this study, we developed a single-molecule desktop sequencer, GenoCare 1600 (GenoCare), which utilizes amplification-free library preparation and two-color sequencing-by-synthesis chemistry, making it more user-friendly compared with previous single-molecule sequencing platforms for clinical use. Using the GenoCare platform, we sequenced an Escherichia coli standard sample and achieved a consensus accuracy exceeding 99.99%. We also evaluated the sequencing performance of this platform in microbial mixtures and coronavirus disease 2019 (COVID-19) samples from throat swabs. Our findings indicate that the GenoCare platform allows for microbial quantitation, sensitive identification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, and accurate detection of virus mutations, as confirmed by Sanger sequencing, demonstrating its remarkable potential in clinical application.

Genome sequencing, assembly, and characterization of Pichia fermentans Z9Y-3 as a non-Saccharomyces yeast with aroma enhancing potential

Pichia fermentans is an important non-Saccharomyces yeast applied in mixed fermentation to enhance fruity and floral traits of wine aroma. In this study, genome sequencing of P. fermentans Z9Y-3 was performed using the PacBio single-molecule real-time sequencing platform, and was further assembled using MECAT. The sequenced genes are annotated by non-redundant protein (Nr), Gene Ontology (GO), Cluster of Orthologous Groups of proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and carbohydrate-active enzymes (CAZymes) database. Results indicated that the genome size was about 13.5 Mb with 42.09% GC content and 4730 protein coding genes. The 95.90% genes were annotated to the non-redundant protein (Nr) database, and then annotated to GO database. The 62.64% genes annotated to the COG database revealed 25 functional groups. The 58.22% genes annotated to the KEGG database revealed 5 groups. The 586 genes were annotated to CAZymes, in which two genes bglH and bglC encoding beta-glucosidase were found and further analyzed its motifs. A terpene and a nonribosomal peptides like secondary metabolic gene clusters were predicted using antiSMASH 7.0.0. This study benefits us to understand physiological and biochemical behavior of P. fermentans Z9Y-3 at a molecular level to better use P. fermentans Z9Y-3 in aroma enhancing winemaking.

Abstract 5301: Detection of arginine posttranslational modifications by single-molecule protein sequencing on the Quantum-Si platform

Abstract Aberrant post-translational modifications (PTMs) on arginine residues, such as methylation and citrullination, are closely linked to oncogenic processes. Detection of these PTMs is challenging with current technology and can be hampered by low abundance and mass differences that are difficult to resolve by mass spectrometry. To overcome these challenges, we developed methods for PTM detection using Quantum-Si's Platinum single-molecule protein sequencing platform. Proteins are sequenced on Quantum-Si's Platinum instrument using our library prep protocol. Briefly, proteins are digested into peptide fragments, conjugated to linkers, then immobilized at the bottom of nanoscale reaction chambers on a semiconductor chip, resulting in exposed N-termini for sequencing. During sequencing, the immobilized peptides are exposed to a solution of dye-labeled N-terminal amino acid (NAA) recognizers that reversibly bind to their cognate NAAs with distinct kinetic properties. Aminopeptidases sequentially remove NAAs to expose subsequent amino acids for recognition. Fluorescence lifetime, intensity, and kinetic data are collected in real time and analyzed to determine primary sequence and PTM content. The data output consists of distinct pulsing regions called recognition segments (RSs). Each RS corresponds to a period of time between aminopeptidase cleavage events, during which an NAA recognizer binds on/off to its exposed target NAA. Chemical modification to a NAA or to a nearby downstream amino acid can modulate recognizer affinity, resulting in a characteristic change in the average pulse duration (PD) during an RS. To demonstrate the detection and differentiation of arginine PTMs, we applied our platform to distinguish between asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) and between citrulline and native arginine residues. Sequencing traces revealed that arginine and ADMA bind the arginine recognizer (PS621) with similar PD, whereas SDMA exhibited no binding. These results indicate that SDMA, in contrast to ADMA, has reduced binding affinity with PS621, providing a clear kinetic difference between these isomeric arginine PTMs. Moreover, citrulline and arginine side chains also exhibited distinguishable kinetic signatures. Citrullination eliminated the N-terminal arginine recognized by PS621 and resulted in a large increase in the median PD of preceding amino acid residues. Taken together, single-molecule protein sequencing offers an alternative approach to detection of arginine PTMs that is not based on m/z, but rather on the kinetic signature of binding between recognizers and N-terminal amino acids. The ability to directly detect arginine PTMs offers potential for biomedical research. We envisage applications using Quantum-Si's platform to key areas of cancer research including biomarker development and drug discovery. Citation Format: Kenneth Skinner, David Kamber, Brian Reed. Detection of arginine posttranslational modifications by single-molecule protein sequencing on the Quantum-Si platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5301.

Evolutional transition of HBV genome during the persistent infection determined by single-molecule real-time sequencing.

Although HBV infection is a serious health issue worldwide, the landscape of HBV genome dynamics in the host has not yet been clarified. This study aimed to determine the continuous genome sequence of each HBV clone using a single-molecule real-time sequencing platform, and clarify the dynamics of structural abnormalities during persistent HBV infection without antiviral therapy. Twenty-five serum specimens were collected from 10 untreated HBV-infected patients. Continuous whole-genome sequencing of each clone was performed using a PacBio Sequel sequencer; the relationship between genomic variations and clinical information was analyzed. The diversity and phylogeny of the viral clones with structural variations were also analyzed. The whole-genome sequences of 797,352 HBV clones were determined. The deletion was the most common structural abnormality and concentrated in the preS/S and C regions. Hepatitis B e antibody (anti-HBe)-negative samples or samples with high alanine aminotransferase levels have significantly diverse deletions than anti-HBe-positive samples or samples with low alanine aminotransferase levels. Phylogenetic analysis demonstrated that various defective and full-length clones evolve independently and form diverse viral populations. Single-molecule real-time long-read sequencing revealed the dynamics of genomic quasispecies during the natural course of chronic HBV infections. Defective viral clones are prone to emerge under the condition of active hepatitis, and several types of defective variants can evolve independently of the viral clones with the full-length genome.

HLA/MHC and KIR characterization in humans and non-human primates using Oxford Nanopore Technologies and Pacific Biosciences sequencing platforms.

The gene products of the HLA/MHC and KIR multigene families are important modulators of the immune system and are associated with health and disease. Characterization of the genes encoding these receptors has been integrated into different biomedical applications, including transplantation and reproduction biology, immune therapies and in fundamental research into disease susceptibility or resistance. Conventional short-read sequencing strategies have shown their value in high throughput typing, but are insufficient to uncover the entire complexity of the highly polymorphic HLA/MHC and KIR gene systems. The implementation of single-molecule and real-time sequencing platforms, offered by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), revolutionized the fields of genomics and transcriptomics. Using fundamentally distinct principles, these platforms generate long-read data that can unwire the plasticity of the HLA/MHC and KIR genes, including high-resolution characterization of genes, alleles, phased haplotypes, transcription levels and epigenetics modification patterns. These insights might have profound clinical relevance, such as improved matching of donors and patients in clinical transplantation, but could also lift disease association studies to a higher level. Even more, a comprehensive characterization may refine animal models in preclinical studies. In this review, the different HLA/MHC and KIR characterization approaches using PacBio and ONT platforms are described and discussed.

High-resolution phylogenetic and population genetic analysis of microbial communities with RoC-ITS

Microbial communities are inter-connected systems of incredible complexity and dynamism that play crucial roles in health, energy, and the environment. To better understand microbial communities and how they respond to change, it is important to know which microbes are present and their relative abundances at the greatest taxonomic resolution possible. Here, we describe a novel protocol (RoC-ITS) that uses the single-molecule Nanopore sequencing platform to assay the composition of microbial communities at the subspecies designation. Using rolling-circle amplification, this methodology produces long-read sequences from a circular construct containing the complete 16S ribosomal gene and the neighboring internally transcribed spacer (ITS). These long reads can be used to generate a high-fidelity circular consensus sequence. Generally, the ribosomal 16S gene provides phylogenetic information down to the species-level, while the much less conserved ITS region contains strain-level information. When linked together, this combination of markers allows for the identification of individual ribosomal units within a specific organism and the assessment of their relative stoichiometry, as well as the ability to monitor subtle shifts in microbial community composition with a single generic assay. We applied RoC-ITS to an artificial microbial community that was also sequenced using the Illumina platform, to assess its accuracy in quantifying the relative abundance and identity of each species.

In-field genetic stock identification of overwintering coho salmon in the Gulf of Alaska: Evaluation of Nanopore sequencing for remote real-time deployment.

Genetic stock identification (GSI) from genotyping‐by‐sequencing of single nucleotide polymorphism (SNP) loci has become the gold standard for stock of origin identification in Pacific salmon. The sequencing platforms currently applied require large batch sizes and multiday processing in specialized facilities to perform genotyping by the thousands. However, recent advances in third‐generation single‐molecule sequencing platforms, such as the Oxford Nanopore minION, provide base calling on portable, pocket‐sized sequencers and promise real‐time, in‐field stock identification of variable batch sizes. Here we evaluate utility and comparability to established GSI platforms of at‐sea stock identification of coho salmon (Oncorhynchus kisutch) using targeted SNP amplicon sequencing on the minION platform during a high‐sea winter expedition to the Gulf of Alaska. As long read sequencers are not optimized for short amplicons, we concatenate amplicons to increase coverage and throughput. Nanopore sequencing at‐sea yielded data sufficient for stock assignment for 50 out of 80 individuals. Nanopore‐based SNP calls agreed with Ion Torrent‐based genotypes in 83.25%, but assignment of individuals to stock of origin only agreed in 61.5% of individuals, highlighting inherent challenges of Nanopore sequencing, such as resolution of homopolymer tracts and indels. However, poor representation of assayed salmon in the queried baseline data set contributed to poor assignment confidence on both platforms. Future improvements will focus on lowering turnaround time and cost, increasing accuracy and throughput, as well as augmentation of the existing baselines. If successfully implemented, Nanopore sequencing will provide an alternative method to the large‐scale laboratory approach by providing mobile small batch genotyping to diverse stakeholders.

Nanopore sequencing of RNA and cDNA molecules in Escherichia coli.

High-throughput sequencing dramatically changed our view of transcriptome architectures and allowed for ground-breaking discoveries in RNA biology. Recently, sequencing of full-length transcripts based on the single-molecule sequencing platform from Oxford Nanopore Technologies (ONT) was introduced and is widely used to sequence eukaryotic and viral RNAs. However, experimental approaches implementing this technique for prokaryotic transcriptomes remain scarce. Here, we present an experimental and bioinformatic workflow for ONT RNA-seq in the bacterial model organism Escherichia coli, which can be applied to any microorganism. Our study highlights critical steps of library preparation and computational analysis and compares the results to gold standards in the field. Furthermore, we comprehensively evaluate the applicability and advantages of different ONT-based RNA sequencing protocols, including direct RNA, direct cDNA, and PCR-cDNA. We find that (PCR)-cDNA-seq offers improved yield and accuracy compared to direct RNA sequencing. Notably, (PCR)-cDNA-seq is suitable for quantitative measurements and can be readily used for simultaneous and accurate detection of transcript 5′ and 3′ boundaries, analysis of transcriptional units, and transcriptional heterogeneity. In summary, based on our comprehensive study, we show nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features. Thereby nanopore RNA-seq holds the potential to become a valuable alternative method for RNA analysis in prokaryotes.

Abstract LB201: Understanding genetic variation in cancer using nanopore targeted sequencing

Abstract Structural variations (SV), a hallmark of genomic instability in cancer can either activate oncogenes or inactivate tumor suppressor genes. SVs tend to be recurrent and have been associated with several cancer types. Next-generation sequencing (NGS) is mostly blind to large SVs, lacking sensitivity with false positive rates up to 89% in SV detection. Long-read, single-molecule sequencing platforms can address larger variations as they typically generate read lengths of tens of thousands of bases and have helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. However, the throughput and coverage offered by whole genome long read sequencing makes it infeasible to conduct large-scale genomic studies.Targeted sequencing significantly improves accuracy and coverage by offering the depth necessary to detect rare alleles in a heterogenous population of cells. Using the nanopore Cas9-targeted sequencing (nCATS), a PCR-free enrichment system from Oxford Nanopore Technologies, we targeted 10 prominent cancer genes in MCF 10A and SK-BR-3 breast cell lines. However, we observed that the number of reads generated for targets longer than 30kb were not sufficient to accurately call SVs. To further enhance this approach, we developed an Affinity-based Cas-9-Mediated Enrichment (ACME) step, that uses HisTag DynabeadsTM to pulldown non-target reads. With ACME we achieved ~ 75-fold enrichment of the BRCA2 region and 35-65x coverage of this ~90 kb target on our panel. We observed an increase in enrichment and coverage of other genes on the panel as well, with enrichment as high as 5000-fold for some genes. While ACME is a biochemical approach, recently, adaptive sequencing or “Read Until”, a computational approach that ‘rejects' non-target reads has been used by a couple of groups for target enrichment. Using UNCALLED, a variation of Read Until, we observed an ~10x coverage of our targets. Recognizing the potential and limitations of both, biochemical and computational approaches, our efforts now are directed towards a combined approach where we expect to achieve a minimum enrichment of 200-fold across all targets. With a 200-fold enrichment of a 3.5Mb target, we should get between 200x - 400x coverage of the target, which should be high enough to detect SVs in as low as 10% of the cells in the sample. This would support large scale SV analysis to help define the landscape of such variants in the population and identify regions of therapeutic or diagnostic interest. Citation Format: Shruti V. Iyer, Melissa Kramer, Sara Goodwin, W. Richard McCombie. Understanding genetic variation in cancer using nanopore targeted sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB201.

Whole-genome sequencing exploitation analysis of non-Saccharomyces yeast Nakazawaea ishiwadae GDMCC 60786 and its physiological characterizations

The genomic and metabolic features of non-Saccharomyces yeast Nakazawaea ishiwadae GDMCC 60786 were investigated using its genome and by analyzing the metabolites of carbon source fermentation. N. ishiwadae GDMCC 60786, which showed resistant to environmental stresses (ethanol, SO2, pH, and NaCl) and a high ethyl acetate-production ability, was classified into the Nakazawaea genus based on the 26S rRNA gene. Complete genome sequences of the strain were obtained using the PacBio single-molecule real-time sequencing platform. The genome size was about 12,088,154 bp with 33.69% GC content. Key enzymes of glycolytic and pyruvate metabolism were identified by genes function annotation. KEGG analysis revealed that this strain possesses phosphoglucomutase, phosphofructokinase, phosphoglycerate kinase, phosphoglycerate mutase, pyruvate decarboxylase, and pyruvate dehydrogenase. Furthermore, combined with fermentation data on various carbon sources (e.g., glucose, fructose, maltose, and sucrose) and different amounts of ethanol and acetic acid, it was found that monosaccharides were more suitable as the carbon source of this strain to produce ethyl acetate. Compared with the control group, 4% (v/v) ethanol was used as supplementary carbon source, the content of ethyl acetate was increased 4 times.

Electrokinetic identification of ribonucleotide monophosphates (rNMPs) using thermoplastic nanochannels

With advances in the design and fabrication of nanofluidic devices during the last decade, there have been a few reports on nucleic acid analysis using nanoscale electrophoresis. The attractive nature of nanofluidics is the unique phenomena associated with this length scale that are not observed using microchip electrophoresis. Many of these effects are surface-related and include electrostatics, surface roughness, van der Waals interactions, hydrogen bonding, and the electric double layer. The majority of reports related to nanoscale electrophoresis have utilized glass-based devices, which are not suitable for broad dissemination into the separation community because of the sophisticated, time consuming, and high-cost fabrication methods required to produce the relevant devices. In this study, we report the use of thermoplastic nanochannels (110 nm x 110 nm, depth x width) for the free solution electrokinetic analysis of ribonucleotide monophosphates (rNMPs). Thermoplastic devices with micro- and nanofluidic networks were fabricated using nanoimprint lithography (NIL) with the structures enclosed via thermal fusion bonding of a cover plate to the fluidic substrate. Unique to this report is that we fabricated devices in cyclic olefin copolymer (COC) that was thermally fusion bonded to a COC cover plate. Results using COC/COC devices were compared to poly(methyl methacrylate), PMMA, devices with a COC cover plate. Our results indicated that at pH = 7.9, the electrophoresis in free solution resulted in an average resolution of the rNMPs >4 (COC/COC device range = 1.94 – 8.88; PMMA/COC device range = 1.4 – 7.8) with some of the rNMPs showing field-dependent electrophoretic mobilities. Baseline separation of the rNMPs was not possible using PMMA- or COC-based microchip electrophoresis. We also found that COC/COC devices could be assembled and UV/O3 activated after device assembly with the dose of the UV/O3 affecting the magnitude of the electroosmotic flow, EOF. In addition, the bond strength between the substrate and cover plate of unmodified COC/COC devices was higher compared to PMMA/COC devices. The large differences in the electrophoretic mobilities of the rNMPs afforded by nanoscale electrophoresis will enable a new single-molecule sequencing platform we envision, which uses molecular-dependent electrophoretic mobilities to identify the constituent rNMPs generated from an intact RNA molecule using a processive exonuclease. With optimized nanoscale electrophoresis, the rNMPs could be identified via mobility matching at an accuracy >99% in both COC/COC and PMMA/COC devices.

Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases.

DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for specific properties gives the opportunity to identify polymerases with different features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to an α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of ∼100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with altered physical properties.

Complete Draft Genome Sequence of Cutibacterium (Propionibacterium) acnes Type Strain ATCC6919

Abstract Objectives: The wide use of antibiotics for acne is the main cause of drug-resistant Cutibacterium acnes. However, the complete genetic sequence reference of C. acnes has not yet been reported. Herein, we report the complete genetic sequence of Cutibacterium acnes type strain ATCC6919. Methods: Whole-genome sequencing of Cutibacterium acnes ATCC6919 was performed using both the Illumina Hiseq4000 and PacBio RSII single-molecule real-time sequencing platforms. The generated sequence reads were de novo assembled using the HGAP3 protocol of single-molecule real-time analysis v.2.3.0 software. The draft genome was annotated and further analyzed by various bioinformatics tools. Results: The total genome length of Cutibacterium acnes ATCC6919 was 2,495,001 bp (60.02% G + C content), with 2,450 coding sequences including 2,358 proteins, 45 tRNAs, 9 rRNAs, and 1 sRNA. The Comprehensive Antibiotic Resistance Database showed that the gene (thy A) found in ATCC6919 was associated with resistance to aminosalicylic acid. Antimicrobial susceptibility testing showed that this isolate was not sensitive to metronidazole and sulfamethoxazole, but was sensitive to all other tested antimicrobials (erythromycin, azithromycin, clarithromycin, tetracycline, minocycline, trimethoprim, levofloxacin, ceftriaxone, and doxycycline). Conclusion: This study reports the complete genome sequence of Cutibacterium acnes ATCC6919, which provides a valuable reference for comparative analysis based on the genomic characteristics and resistance gene diversity of Cutibacterium acnes.

Evaluation of Single-Molecule Sequencing Technologies for Structural Variant Detection in Two Swedish Human Genomes.

Long-read single molecule sequencing is increasingly used in human genomics research, as it allows to accurately detect large-scale DNA rearrangements such as structural variations (SVs) at high resolution. However, few studies have evaluated the performance of different single molecule sequencing platforms for SV detection in human samples. Here we performed Oxford Nanopore Technologies (ONT) whole-genome sequencing of two Swedish human samples (average 32× coverage) and compared the results to previously generated Pacific Biosciences (PacBio) data for the same individuals (average 66× coverage). Our analysis inferred an average of 17k and 23k SVs from the ONT and PacBio data, respectively, with a majority of them overlapping with an available multi-platform SV dataset. When comparing the SV calls in the two Swedish individuals, we find a higher concordance between ONT and PacBio SVs detected in the same individual as compared to SVs detected by the same technology in different individuals. Downsampling of PacBio reads, performed to obtain similar coverage levels for all datasets, resulted in 17k SVs per individual and improved overlap with the ONT SVs. Our results suggest that ONT and PacBio have a similar performance for SV detection in human whole genome sequencing data, and that both technologies are feasible for population-scale studies.

Abstract 1360: Understanding genetic variation in cancer using targeted nanopore long read sequencing

Abstract Structural variations (SV), a hallmark of genomic instability in cancer can either activate oncogenes or inactivate tumor suppressor genes. SVs tend to be recurrent and have been associated with several cancer types. They are pervasive in nature however, next-generation sequencing (NGS) is mostly blind to SVs. They lack sensitivity and exhibit very high false positive rates (up to 89%) in SV detection. Long-read, single-molecule sequencing platforms like Pacific Biosciences and Oxford Nanopore Technologies (ONT) can address larger variations as they typically generate read lengths of tens of thousands of bases and have helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. However, the throughput and coverage offered by whole genome long read sequencing makes it infeasible to conduct large-scale genomic studies. Targeted sequencing significantly improves accuracy and coverage by offering the depth necessary to detect rare alleles in a heterogenous population of cells. It will facilitate large population SV analysis, which will help define the landscape of such variants in the population and help identify regions of therapeutic or diagnostic interest. However, a lack of efficient long-read compatible targeting techniques makes it difficult to study specific regions of interest on existing long-read platforms. To address this, we are evaluating CRISPR/Cas-based systems of targeted long-read sequencing to enrich for specific regions of the cancer genome (panel of 12 genes, including BRCA1 and BRCA2) in two breast cell lines – MCF 10A and SK-BR-3. Using the ONT Cas9-mediated PCR-free enrichment system we targeted 12 genes of interest in two breast cell lines – MCF 10A and SK-BR-3. Initially, we used guides that targeted a 200kb region around the BRCA1 gene in SK-BR-3 and generated a 198kb read spanning the entire BRCA1 region. However, the number of reads on target were not sufficient to accurately call SVs. To address the issue of low on-target reads, we included an Affinity-based Cas-9-Mediated Enrichment (ACME) step, that uses a HisTag-based isolation and pulldown of background/non-target reads. By targeting all 11 genes together in a single reaction, our ACME worked extremely well, since each reaction now had over 10 times as many guides as opposed to targeting a single gene per reaction. This gave us more cut sites and Cas9-bound non-targets, increasing the enrichment efficiency of the HisTag pulldown, giving us a 75-fold enrichment of the BRCA2 region and close to 100x coverage of the entire 95kb target. We observed an increase in enrichment and coverage of the other genes on the panel as well, with enrichment as high as 4000-fold for some genes. Our goal is to develop a long-read breast cancer panel to facilitate large population SV analysis, which will help define the landscape of such variants in the population and identify regions of therapeutic or diagnostic interest. Citation Format: Shruti V. Iyer, Sara Goodwin, Melissa Kramer, W. Richard McCombie. Understanding genetic variation in cancer using targeted nanopore long read sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1360.

A chromosome-level genome assembly of the wild rice Oryza rufipogon facilitates tracing the origins of Asian cultivated rice.

Oryza rufipogon Griff. is a wild progenitor of the Asian cultivated rice Oryza sativa. To better understand the genomic diversity of the wild rice, high-quality reference genomes of O. rufipogon populations are needed, which also facilitate utilization of the wild genetic resources in rice breeding. In this study, we generated a chromosome-level genome assembly of O. rufipogon using a combination of short-read sequencing, single-molecule sequencing, BioNano and Hi-C platforms. The genome sequence (399.8 Mb) was assembled into 46 scaffolds on the 12 chromosomes, with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb, respectively. The genome contains 36,520 protein-coding genes, and 49.37% of the genome consists of repetitive elements. The genome has strong synteny with those of the O. sativa subspecies indica and japonica, but containing some large structural variations. Evolutionary analysis unveiled the polyphyletic origins of O. sativa, in which the japonica and indica genome formations involved different divergent O. rufipogon (including O. nivara) lineages, accompanied by introgression of genomic regions between japonica and indica. This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O. sativa subspecies, and valuable information for basic research and rice breeding.

Profiling of Human Gut Virome with Oxford Nanopore Technology

Human gut virome play critical roles in maintaining gut microbial composition and functionality, as well as host physiology and immunology. Yet, there are insufficient amount of studies on this topic mainly due to methodological limitations, including enrichment of viruses (phages and host viruses) as well as short read-length from current sequencing technology. Here we developed a full working protocol for analyzing human gut virome using physical enrichment, reverse transcription and random amplification, and eventually the state-of-art single-molecule real-time sequencing (SMRT) platform of Oxford Nanopore Technology (ONT). We demonstrate that sequencing viral DNA directly, or viral cDNA/DNA after amplification using ONT achieves much longer reads and provides more information regarding virome diversity, many of the virome sequences do not have match in current databases. Moreover, direct DNA sequencing of virome provides first insights into the epigenetic modifications on phages, where signals of methylations can be directly detected. Our study demonstrates that progressing sequencing technology and bioinformatic improvements will bring more knowledge into virome composition, diversity and potentially their important functions.

Rapid high-resolution HLA genotyping by MinION Oxford nanopore sequencing for deceased donor organ allocation.

Recently, HLA epitopes on donor HLA molecules have been shown to be important in the success of solid organ transplantation. However, these epitopes can only be defined using high-resolution typing results of which are often not available prior to deceased donor allocation. The ability to perform high-resolution typing at all HLA loci for deceased organ donor allocation prior to transplantation would have major clinical benefits, in particular for highly sensitised recipients. We, therefore, developed a rapid high-resolution next generation sequencing (NGS) HLA typing (ONT-Rapid HR HLA) method for on-call deceased donor allocation using the AllType 11 loci single-tube assay (OneLambda Inc), modified in-house to reduce polymerase chain reaction amplification time, and the Oxford Nanopore single-molecule sequencing platform on the Flongle flow cell. The ONT-Rapid HR HLA method was validated on 42 samples previously typed by current on-call sequence-specific oligonucleotide (HistoSpot) and NGS methods (AllType/Ion Torrent). High-resolution typing obtained using the ONT-Rapid HR HLA typing method was 100% concordant with both the current SSO and NGS methods, and in some cases, obtained higher resolution than either of the current methods. The rapid ONT-Rapid HR HLA typing method was able to obtain these typing results at all loci in 4 to 4.5 hours. The novel ONT-Rapid HR HLA typing method is the first reported NGS HLA typing method utilised for deceased donor allocation. The ability to provide high-resolution HLA typing on deceased donors before implantation will in the future allow improvements in matching, which will ultimately provide clinical benefits to patients.

SERS discrimination of single DNA bases in single oligonucleotides by electro-plasmonic trapping

Surface-enhanced Raman spectroscopy (SERS) sensing of DNA bases by plasmonic nanopores could pave a way to novel methods for DNA analyses and new generation single-molecule sequencing platforms. The SERS discrimination of single DNA bases depends critically on the time that a DNA strand resides within the plasmonic hot spot. In fact, DNA molecules flow through the nanopores so rapidly that the SERS signals collected are not sufficient for single-molecule analysis. Here, we report an approach to control the residence time of molecules in the hot spot by an electro-plasmonic trapping effect. By directly adsorbing molecules onto a gold nanoparticle and then trapping the single nanoparticle in a plasmonic nanohole up to several minutes, we demonstrate single-molecule SERS detection of all four DNA bases as well as discrimination of single nucleobases in a single oligonucleotide. Our method can be extended easily to label-free sensing of single-molecule amino acids and proteins.

The complete genome sequence of Ethanoligenens harbinense reveals the metabolic pathway of acetate-ethanol fermentation: A novel understanding of the principles of anaerobic biotechnology

Ethanol-type fermentation is one of three main fermentation types in the acidogenesis of anaerobic treatment systems. Non-spore-forming Ethanoligenens is as a typical genus capable of ethanol-type fermentation in mixed culture (i.e. acetate-ethanol fermentation). This genus can produce ethanol, acetate, CO2, and H2 using carbohydrates, and has application potential in anaerobic bioprocesses. Here, the complete genome sequences and methylome of Ethanoligenens harbinense strains with different autoaggregative and coaggregative abilities were obtained using the PacBio single-molecule real-time sequencing platform. The genome size of E. harbinense strains was about 2.97–3.10 Mb with 55.5% G+C content. 3020–3153 genes were annotated, most of which were methylated at specific sites or motifs. The methylation types included 6mA, 4mC, and unknown types. Comparative genomic analysis demonstrated low levels of genetic similarity between E. harbinense and other well-known hydrogen-producing bacteria (i.e., Clostridium and Thermoanaerobacter) in phylogenesis. Hydrogen production of E. harbinense was catalyzed by genes that encode [FeFe]‑hydrogenases and that were synthesized by three maturases of [FeFe]-H2ase. The metabolic mechanism of H2-ethanol co-production fermentation, catalyzed by pyruvate ferredoxin oxidoreductase was proposed. This study provides genetic and evolutionary information of a model genus for the further investigation of the metabolic pathway and regulatory network of ethanol-type fermentation and anaerobic bioprocesses for waste or wastewater treatment.