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1
Pradeep, Chaithra, Dharam Nandan, Arya A. Das, and Dinesh Velayutham. "Comparative Transcriptome Profiling of Disruptive Technology, Single- Molecule Direct RNA Sequencing." Current Bioinformatics 15, no. 2 (March 10, 2020): 165–72. http://dx.doi.org/10.2174/1574893614666191017154427.
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Background: The standard approach for transcriptomic profiling involves high throughput short-read sequencing technology, mainly dominated by Illumina. However, the short reads have limitations in transcriptome assembly and in obtaining full-length transcripts due to the complex nature of transcriptomes with variable length and multiple alternative spliced isoforms. Recent advances in long read sequencing by the Oxford Nanopore Technologies (ONT) offered both cDNA as well as direct RNA sequencing and has brought a paradigm change in the sequencing technology to greatly improve the assembly and expression estimates. ONT enables molecules to be sequenced without fragmentation resulting in ultra-long read length enabling the entire genes and transcripts to be fully characterized. The direct RNA sequencing method, in addition, circumvents the reverse transcription and amplification steps. Objective: In this study, RNA sequencing methods were assessed by comparing data from Illumina (ILM), ONT cDNA (OCD) and ONT direct RNA (ODR). Methods: The sensitivity & specificity of the isoform detection was determined from the data generated by Illumina, ONT cDNA and ONT direct RNA sequencing technologies using Saccharomyces cerevisiae as model. Comparative studies were conducted with two pipelines to detect the isoforms, novel genes and variable gene length. Results: Mapping metrics and qualitative profiles for different pipelines are presented to understand these disruptive technologies. The variability in sequencing technology and the analysis pipeline were studied.
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Weirather, Jason L., Mariateresa de Cesare, Yunhao Wang, Paolo Piazza, Vittorio Sebastiano, Xiu-Jie Wang, David Buck, and Kin Fai Au. "Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis." F1000Research 6 (February 3, 2017): 100. http://dx.doi.org/10.12688/f1000research.10571.1.
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Background: Given the demonstrated utility of Third Generation Sequencing [Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)] long reads in many studies, a comprehensive analysis and comparison of their data quality and applications is in high demand. Methods: Based on the transcriptome sequencing data from human embryonic stem cells, we analyzed multiple data features of PacBio and ONT, including error pattern, length, mappability and technical improvements over previous platforms. We also evaluated their application to transcriptome analyses, such as isoform identification and quantification and characterization of transcriptome complexity, by comparing the performance of PacBio, ONT and their corresponding Hybrid-Seq strategies (PacBio+Illumina and ONT+Illumina). Results: PacBio shows overall better data quality, while ONT provides a higher yield. As with data quality, PacBio performs marginally better than ONT in most aspects for both long reads only and Hybrid-Seq strategies in transcriptome analysis. In addition, Hybrid-Seq shows superior performance over long reads only in most transcriptome analyses. Conclusions: Both PacBio and ONT sequencing are suitable for full-length single-molecule transcriptome analysis. As this first use of ONT reads in a Hybrid-Seq analysis has shown, both PacBio and ONT can benefit from a combined Illumina strategy. The tools and analytical methods developed here provide a resource for future applications and evaluations of these rapidly-changing technologies.
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Weirather, Jason L., Mariateresa de Cesare, Yunhao Wang, Paolo Piazza, Vittorio Sebastiano, Xiu-Jie Wang, David Buck, and Kin Fai Au. "Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis." F1000Research 6 (June 19, 2017): 100. http://dx.doi.org/10.12688/f1000research.10571.2.
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Background: Given the demonstrated utility of Third Generation Sequencing [Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)] long reads in many studies, a comprehensive analysis and comparison of their data quality and applications is in high demand. Methods: Based on the transcriptome sequencing data from human embryonic stem cells, we analyzed multiple data features of PacBio and ONT, including error pattern, length, mappability and technical improvements over previous platforms. We also evaluated their application to transcriptome analyses, such as isoform identification and quantification and characterization of transcriptome complexity, by comparing the performance of size-selected PacBio, non-size-selected ONT and their corresponding Hybrid-Seq strategies (PacBio+Illumina and ONT+Illumina). Results: PacBio shows overall better data quality, while ONT provides a higher yield. As with data quality, PacBio performs marginally better than ONT in most aspects for both long reads only and Hybrid-Seq strategies in transcriptome analysis. In addition, Hybrid-Seq shows superior performance over long reads only in most transcriptome analyses. Conclusions: Both PacBio and ONT sequencing are suitable for full-length single-molecule transcriptome analysis. As this first use of ONT reads in a Hybrid-Seq analysis has shown, both PacBio and ONT can benefit from a combined Illumina strategy. The tools and analytical methods developed here provide a resource for future applications and evaluations of these rapidly-changing technologies.
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Fatima, Nazeefa, Anna Petri, Ulf Gyllensten, Lars Feuk, and Adam Ameur. "Evaluation of Single-Molecule Sequencing Technologies for Structural Variant Detection in Two Swedish Human Genomes." Genes 11, no. 12 (November 30, 2020): 1444. http://dx.doi.org/10.3390/genes11121444.
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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.
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Zhang, Pengfei, Dike Jiang, Yin Wang, Xueping Yao, Yan Luo, and Zexiao Yang. "Comparison of De Novo Assembly Strategies for Bacterial Genomes." International Journal of Molecular Sciences 22, no. 14 (July 17, 2021): 7668. http://dx.doi.org/10.3390/ijms22147668.
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(1) Background: Short-read sequencing allows for the rapid and accurate analysis of the whole bacterial genome but does not usually enable complete genome assembly. Long-read sequencing greatly assists with the resolution of complex bacterial genomes, particularly when combined with short-read Illumina data. However, it is not clear how different assembly strategies affect genomic accuracy, completeness, and protein prediction. (2) Methods: we compare different assembly strategies for Haemophilus parasuis, which causes Glässer’s disease, characterized by fibrinous polyserositis and arthritis, in swine by using Illumina sequencing and long reads from the sequencing platforms of either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio). (3) Results: Assembly with either PacBio or ONT reads, followed by polishing with Illumina reads, facilitated high-quality genome reconstruction and was superior to the long-read-only assembly and hybrid-assembly strategies when evaluated in terms of accuracy and completeness. An equally excellent method was correction with Homopolish after the ONT-only assembly, which had the advantage of avoiding hybrid sequencing with Illumina. Furthermore, by aligning transcripts to assembled genomes and their predicted CDSs, the sequencing errors of the ONT assembly were mainly indels that were generated when homopolymer regions were sequenced, thus critically affecting protein prediction. Polishing can fill indels and correct mistakes. (4) Conclusions: The assembly of bacterial genomes can be directly achieved by using long-read sequencing techniques. To maximize assembly accuracy, it is essential to polish the assembly with homologous sequences of related genomes or sequencing data from short-read technology.
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Dumschott, Kathryn, Maximilian H.-W. Schmidt, Harmeet Singh Chawla, Rod Snowdon, and Björn Usadel. "Oxford Nanopore sequencing: new opportunities for plant genomics?" Journal of Experimental Botany 71, no. 18 (May 27, 2020): 5313–22. http://dx.doi.org/10.1093/jxb/eraa263.
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Abstract DNA sequencing was dominated by Sanger’s chain termination method until the mid-2000s, when it was progressively supplanted by new sequencing technologies that can generate much larger quantities of data in a shorter time. At the forefront of these developments, long-read sequencing technologies (third-generation sequencing) can produce reads that are several kilobases in length. This greatly improves the accuracy of genome assemblies by spanning the highly repetitive segments that cause difficulty for second-generation short-read technologies. Third-generation sequencing is especially appealing for plant genomes, which can be extremely large with long stretches of highly repetitive DNA. Until recently, the low basecalling accuracy of third-generation technologies meant that accurate genome assembly required expensive, high-coverage sequencing followed by computational analysis to correct for errors. However, today’s long-read technologies are more accurate and less expensive, making them the method of choice for the assembly of complex genomes. Oxford Nanopore Technologies (ONT), a third-generation platform for the sequencing of native DNA strands, is particularly suitable for the generation of high-quality assemblies of highly repetitive plant genomes. Here we discuss the benefits of ONT, especially for the plant science community, and describe the issues that remain to be addressed when using ONT for plant genome sequencing.
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Maestri, Simone, Maria Giovanna Maturo, Emanuela Cosentino, Luca Marcolungo, Barbara Iadarola, Elisabetta Fortunati, Marzia Rossato, and Massimo Delledonne. "A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings." International Journal of Molecular Sciences 21, no. 23 (December 1, 2020): 9177. http://dx.doi.org/10.3390/ijms21239177.
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The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the APOE locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing.
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Sutton, John M., Joshua D. Millwood, A. Case McCormack, and Janna L. Fierst. "Optimizing experimental design for genome sequencing and assembly with Oxford Nanopore Technologies." Gigabyte 2021 (July 13, 2021): 1–26. http://dx.doi.org/10.46471/gigabyte.27.
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High quality reference genome sequences are the core of modern genomics. Oxford Nanopore Technologies (ONT) produces inexpensive DNA sequences, but has high error rates, which make sequence assembly and analysis difficult as genome size and complexity increases. Robust experimental design is necessary for ONT genome sequencing and assembly, but few studies have addressed eukaryotic organisms. Here, we present novel results using simulated and empirical ONT and DNA libraries to identify best practices for sequencing and assembly for several model species. We find that the unique error structure of ONT libraries causes errors to accumulate and assembly statistics plateau as sequence depth increases. High-quality assembled eukaryotic sequences require high-molecular-weight DNA extractions that increase sequence read length, and computational protocols that reduce error through pre-assembly correction and read selection. Our quantitative results will be helpful for researchers seeking guidance for de novo assembly projects.
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Volden, Roger, Theron Palmer, Ashley Byrne, Charles Cole, Robert J. Schmitz, Richard E. Green, and Christopher Vollmers. "Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA." Proceedings of the National Academy of Sciences 115, no. 39 (September 10, 2018): 9726–31. http://dx.doi.org/10.1073/pnas.1806447115.
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High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples.
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Abeynayake, Shamila Weerakoon, Sonia Fiorito, Adrian Dinsdale, Mark Whattam, Bill Crowe, Kate Sparks, Paul Richard Campbell, and Cherie Gambley. "A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes." Genes 12, no. 8 (July 27, 2021): 1138. http://dx.doi.org/10.3390/genes12081138.
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The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border.
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Carter, Jean-Michel, and Shobbir Hussain. "Robust long-read native DNA sequencing using the ONT CsgG Nanopore system." Wellcome Open Research 2 (April 6, 2017): 23. http://dx.doi.org/10.12688/wellcomeopenres.11246.1.
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Background: The ability to obtain long read lengths during DNA sequencing has several potentially important practical applications. Especially long read lengths have been reported using the Nanopore sequencing method, currently commercially available from Oxford Nanopore Technologies (ONT). However, early reports have demonstrated only limited levels of combined throughput and sequence accuracy. Recently, ONT released a new CsgG pore sequencing system as well as a 250b/s translocation chemistry with potential for improvements. Methods: We made use of such components on ONTs miniature ‘MinION’ device and sequenced native genomic DNA obtained from the near haploid cancer cell line HAP1. Analysis of our data was performed utilising recently described computational tools tailored for nanopore/long-read sequencing outputs, and here we present our key findings. Results: From a single sequencing run, we obtained ~240,000 high-quality mapped reads, comprising a total of ~2.3 billion bases. A mean read length of 9.6kb and an N50 of ~17kb was achieved, while sequences mapped to reference with a mean identity of 85%. Notably, we obtained ~68X coverage of the mitochondrial genome and were able to achieve a mean consensus identity of 99.8% for sequenced mtDNA reads. Conclusions: With improved sequencing chemistries already released and higher-throughput instruments in the pipeline, this early study suggests that ONT CsgG-based sequencing may be a useful option for potential practical long-read applications.
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Carter, Jean-Michel, and Shobbir Hussain. "Robust long-read native DNA sequencing using the ONT CsgG Nanopore system." Wellcome Open Research 2 (May 18, 2017): 23. http://dx.doi.org/10.12688/wellcomeopenres.11246.2.
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Background: The ability to obtain long read lengths during DNA sequencing has several potentially important practical applications. Especially long read lengths have been reported using the Nanopore sequencing method, currently commercially available from Oxford Nanopore Technologies (ONT). However, early reports have demonstrated only limited levels of combined throughput and sequence accuracy. Recently, ONT released a new CsgG pore sequencing system as well as a 250b/s translocation chemistry with potential for improvements. Methods: We made use of such components on ONTs miniature ‘MinION’ device and sequenced native genomic DNA obtained from the near haploid cancer cell line HAP1. Analysis of our data was performed utilising recently described computational tools tailored for nanopore/long-read sequencing outputs, and here we present our key findings. Results: From a single sequencing run, we obtained ~240,000 high-quality mapped reads, comprising a total of ~2.3 billion bases. A mean read length of 9.6kb and an N50 of ~17kb was achieved, while sequences mapped to reference with a mean identity of 85%. Notably, we obtained ~68X coverage of the mitochondrial genome and were able to achieve a mean consensus identity of 99.8% for sequenced mtDNA reads. Conclusions: With improved sequencing chemistries already released and higher-throughput instruments in the pipeline, this early study suggests that ONT CsgG-based sequencing may be a useful option for potential practical long-read applications with relevance to complex genomes.
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Carter, Jean-Michel, and Shobbir Hussain. "Robust long-read native DNA sequencing using the ONT CsgG Nanopore system." Wellcome Open Research 2 (August 30, 2018): 23. http://dx.doi.org/10.12688/wellcomeopenres.11246.3.
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Background: The ability to obtain long read lengths during DNA sequencing has several potentially important practical applications. Especially long read lengths have been reported using the Nanopore sequencing method, currently commercially available from Oxford Nanopore Technologies (ONT). However, early reports have demonstrated only limited levels of combined throughput and sequence accuracy. Recently, ONT released a new CsgG pore sequencing system as well as a 250b/s translocation chemistry with potential for improvements. Methods: We made use of such components on ONTs miniature ‘MinION’ device and sequenced native genomic DNA obtained from the near haploid cancer cell line HAP1. Analysis of our data was performed utilising recently described computational tools tailored for nanopore/long-read sequencing outputs, and here we present our key findings. Results: From a single sequencing run, we obtained ~240,000 high-quality mapped reads, comprising a total of ~2.3 billion bases. A mean read length of 9.6kb and an N50 of ~17kb was achieved, while sequences mapped to reference with a mean identity of 85%. Notably, we obtained ~68X coverage of the mitochondrial genome and were able to achieve a mean consensus identity of 99.8% for sequenced mtDNA reads. Conclusions: With improved sequencing chemistries already released and higher-throughput instruments in the pipeline, this early study suggests that ONT CsgG-based sequencing may be a useful option for potential practical long-read applications with relevance to complex genomes.
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Fellers, John P., Christian Webb, Madison C. Fellers, Jessica Shoup Rupp, and Erick De Wolf. "Wheat Virus Identification Within Infected Tissue Using Nanopore Sequencing Technology." Plant Disease 103, no. 9 (September 2019): 2199–203. http://dx.doi.org/10.1094/pdis-09-18-1700-re.
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Viral diseases are a limiting factor to wheat production. Viruses are difficult to diagnose in the early stages of disease development and are often confused with nutrient deficiencies or other abiotic problems. Immunological methods are useful to identify viruses, but specific antibodies may not be available or require high virus titer for detection. In 2015 and 2017, wheat plants containing Wheat streak mosaic virus (WSMV) resistance gene, Wsm2, were found to have symptoms characteristic of WSMV. Serologically, WSMV was detected in all four samples. Additionally, High Plains wheat mosaic virus (HPWMoV) was also detected in one of the samples. Barley yellow dwarf virus (BYDV) was not detected, and a detection kit was not readily available for Triticum mosaic virus (TriMV). Initially, cDNA cloning and Sanger sequencing were used to determine the presence of WSMV; however, the process was time-consuming and expensive. Subsequently, cDNA from infected wheat tissue was sequenced with single-strand, Oxford Nanopore sequencing technology (ONT). ONT was able to confirm the presence of WSMV. Additionally, TriMV was found in all of the samples and BYDV in three of the samples. Deep coverage sequencing of full-length, single-strand WSMV revealed variation compared with the WSMV Sidney-81 reference strain and may represent new variants which overcome Wsm2. These results demonstrate that ONT can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants.
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Jejaroenpun, Piroon, Thidathip Wongsurawat, Annick DeLoose, David Ussery, Intawat Nookaew, J. D. Day, and Analiz Rodriguez. "GENE-18. TRANSCRIPTOME-WIDE ANALYSIS USING NANOPORE THIRD GENERATION SEQUENCING IN A RAT GLIOBLASTOMA MODEL: PROOF OF PRINCIPLE." Neuro-Oncology 21, Supplement_6 (November 2019): vi101. http://dx.doi.org/10.1093/neuonc/noz175.420.
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Abstract The RNA sequencing (RNA-Seq) technique is now routinely used to quantitatively explore genome-wide expression by various research fields including cancer research. The most common RNA-seq methodology produce billions of short-read sequencing in the range of 100–600 base pairs, from which it is occasionally difficult to reconstruct isoform-level transcriptome and fusion genes. The limitations of the short-reads can be overcome by using third-generation sequencing technologies, such as Oxford Nanopore Technologies (ONT). This study aims to perform full-length cDNA sequencing using ONT platform and investigate the abilities of ONT in (1) identifying differential gene expression, (2) detecting differential transcript isoform usage, and (3) detecting fusion genes. To do these methods, CNS-1 cells were implanted into the frontal lobes of three Lewis rats. The CNS-1 model is a histocompatible astrocytoma cell line with an invasive pattern mimicking glioblastoma (GBM). After two weeks of transplantation, the transplanted tumors and the normal brain on the other side were collected as matched normal-tumor pairs. Total RNA extracted from the samples were subjected to the full-length cDNA sequencing on a portable MinION sequencer. In tumors samples, 615 genes involved in cell cycle were upregulated, whereas 1067 genes involved in neurological functions were downregulated. Finally, we could identify differential transcript isoform expression and fusion genes from the matched normal-tumor pairs. Overall, full-length sequencing of the cDNA molecules permitted a detailed characterization of the differential gene expression, the isoform complexity, and fusion genes. In the near future, we will use these methods on human samples.
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Udaondo, Zulema, Kanchana Sittikankaew, Tanaporn Uengwetwanit, Thidathip Wongsurawat, Chutima Sonthirod, Piroon Jenjaroenpun, Wirulda Pootakham, Nitsara Karoonuthaisiri, and Intawat Nookaew. "Comparative Analysis of PacBio and Oxford Nanopore Sequencing Technologies for Transcriptomic Landscape Identification of Penaeus monodon." Life 11, no. 8 (August 23, 2021): 862. http://dx.doi.org/10.3390/life11080862.
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With the advantages that long-read sequencing platforms such as Pacific Biosciences (Menlo Park, CA, USA) (PacBio) and Oxford Nanopore Technologies (Oxford, UK) (ONT) can offer, various research fields such as genomics and transcriptomics can exploit their benefits. Selecting an appropriate sequencing platform is undoubtedly crucial for the success of the research outcome, thus there is a need to compare these long-read sequencing platforms and evaluate them for specific research questions. This study aims to compare the performance of PacBio and ONT platforms for transcriptomic analysis by utilizing transcriptome data from three different tissues (hepatopancreas, intestine, and gonads) of the juvenile black tiger shrimp, Penaeus monodon. We compared three important features: (i) main characteristics of the sequencing libraries and their alignment with the reference genome, (ii) transcript assembly features and isoform identification, and (iii) correlation of the quantification of gene expression levels for both platforms. Our analyses suggest that read-length bias and differences in sequencing throughput are highly influential factors when using long reads in transcriptome studies. These comparisons can provide a guideline when designing a transcriptome study utilizing these two long-read sequencing technologies.
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Heikema, Astrid P., Deborah Horst-Kreft, Stefan A. Boers, Rick Jansen, Saskia D. Hiltemann, Willem de Koning, Robert Kraaij, et al. "Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota." Genes 11, no. 9 (September 21, 2020): 1105. http://dx.doi.org/10.3390/genes11091105.
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Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies—ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies—ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies—ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable.
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Hardy, Alexis, Mélody Matelot, Amandine Touzeau, Christophe Klopp, Céline Lopez-Roques, Sandra Duharcourt, and Matthieu Defrance. "DNAModAnnot: a R toolbox for DNA modification filtering and annotation." Bioinformatics 37, no. 17 (January 20, 2021): 2738–40. http://dx.doi.org/10.1093/bioinformatics/btab032.
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Abstract Motivation Long-read sequencing technologies can be employed to detect and map DNA modifications at the nucleotide resolution on a genome-wide scale. However, published software packages neglect the integration of genomic annotation and comprehensive filtering when analyzing patterns of modified bases detected using Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) data. Here, we present DNA Modification Annotation (DNAModAnnot), a R package designed for the global analysis of DNA modification patterns using adapted filtering and visualization tools. Results We tested our package using PacBio sequencing data to analyze patterns of the 6-methyladenine (6mA) in the ciliate Paramecium tetraurelia, in which high 6mA amounts were previously reported. We found P. tetraurelia 6mA genome-wide distribution to be similar to other ciliates. We also performed 5-methylcytosine (5mC) analysis in human lymphoblastoid cells using ONT data and confirmed previously known patterns of 5mC. DNAModAnnot provides a toolbox for the genome-wide analysis of different DNA modifications using PacBio and ONT long-read sequencing data. Availability and implementation DNAModAnnot is distributed as a R package available via GitHub (https://github.com/AlexisHardy/DNAModAnnot). Supplementary information Supplementary data are available at Bioinformatics online.
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Zhang, Shoudong, Runsheng Li, Li Zhang, Shengjie Chen, Min Xie, Liu Yang, Yiji Xia, Christine H. Foyer, Zhongying Zhao, and Hon-Ming Lam. "New insights into Arabidopsis transcriptome complexity revealed by direct sequencing of native RNAs." Nucleic Acids Research 48, no. 14 (July 11, 2020): 7700–7711. http://dx.doi.org/10.1093/nar/gkaa588.
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Abstract Arabidopsis thaliana transcriptomes have been extensively studied and characterized under different conditions. However, most of the current ‘RNA-sequencing’ technologies produce a relatively short read length and demand a reverse-transcription step, preventing effective characterization of transcriptome complexity. Here, we performed Direct RNA Sequencing (DRS) using the latest Oxford Nanopore Technology (ONT) with exceptional read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been substantially under-estimated. The ONT direct RNA sequencing identified novel transcript isoforms at both the vegetative (14-day old seedlings, stage 1.04) and reproductive stages (stage 6.00–6.10) of development. Using in-house software called TrackCluster, we determined alternative transcription initiation (ATI), alternative polyadenylation (APA), alternative splicing (AS), and fusion transcripts. More than 38 500 novel transcript isoforms were identified, including six categories of fusion-transcripts that may result from differential RNA processing mechanisms. Aided by the Tombo algorithm, we found an enrichment of m5C modifications in the mobile mRNAs, consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT DRS offers an advantage in the identification and functional characterization of novel RNA isoforms and RNA base modifications, significantly improving annotation of the A. thaliana genome.
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Liefting, Lia W., David W. Waite, and Jeremy R. Thompson. "Application of Oxford Nanopore Technology to Plant Virus Detection." Viruses 13, no. 8 (July 22, 2021): 1424. http://dx.doi.org/10.3390/v13081424.
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The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT’s feasibility as a valuable component to the diagnostician’s toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.
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Alili, Rohia, Eugeni Belda, Phuong Le, Thierry Wirth, Jean-Daniel Zucker, Edi Prifti, and Karine Clément. "Exploring Semi-Quantitative Metagenomic Studies Using Oxford Nanopore Sequencing: A Computational and Experimental Protocol." Genes 12, no. 10 (September 25, 2021): 1496. http://dx.doi.org/10.3390/genes12101496.
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The gut microbiome plays a major role in chronic diseases, of which several are characterized by an altered composition and diversity of bacterial communities. Large-scale sequencing projects allowed for characterizing the perturbations of these communities. However, translating these discoveries into clinical applications remains a challenge. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed. Here, we propose a computational and experimental protocol for whole-genome semi-quantitative metagenomic studies of human gut microbiome with Oxford Nanopore sequencing technology (ONT) that could be applied to other microbial ecosystems. We developed a bioinformatics protocol to analyze ONT sequences taxonomically and functionally and optimized preanalytic protocols, including stool collection and DNA extraction methods to maximize read length. This is a critical parameter for the sequence alignment and classification. Our protocol was evaluated using simulations of metagenomic communities, which reflect naturally occurring compositional variations. Next, we validated both protocols using stool samples from a bariatric surgery cohort, sequenced with ONT, Illumina, and SOLiD technologies. Results revealed similar diversity and microbial composition profiles. This protocol can be implemented in a clinical or research setting, bringing rapid personalized whole-genome profiling of target microbiome species.
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Fukasawa, Yoshinori, Luca Ermini, Hai Wang, Karen Carty, and Min-Sin Cheung. "LongQC: A Quality Control Tool for Third Generation Sequencing Long Read Data." G3: Genes|Genomes|Genetics 10, no. 4 (February 10, 2020): 1193–96. http://dx.doi.org/10.1534/g3.119.400864.
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We propose LongQC as an easy and automated quality control tool for genomic datasets generated by third generation sequencing (TGS) technologies such as Oxford Nanopore technologies (ONT) and SMRT sequencing from Pacific Bioscience (PacBio). Key statistics were optimized for long read data, and LongQC covers all major TGS platforms. LongQC processes and visualizes those statistics automatically and quickly.
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Mohamed, Mourdas, Nguyet Thi-Minh Dang, Yuki Ogyama, Nelly Burlet, Bruno Mugat, Matthieu Boulesteix, Vincent Mérel, et al. "A Transposon Story: From TE Content to TE Dynamic Invasion of Drosophila Genomes Using the Single-Molecule Sequencing Technology from Oxford Nanopore." Cells 9, no. 8 (July 25, 2020): 1776. http://dx.doi.org/10.3390/cells9081776.
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Transposable elements (TEs) are the main components of genomes. However, due to their repetitive nature, they are very difficult to study using data obtained with short-read sequencing technologies. Here, we describe an efficient pipeline to accurately recover TE insertion (TEI) sites and sequences from long reads obtained by Oxford Nanopore Technology (ONT) sequencing. With this pipeline, we could precisely describe the landscapes of the most recent TEIs in wild-type strains of Drosophila melanogaster and Drosophila simulans. Their comparison suggests that this subset of TE sequences is more similar than previously thought in these two species. The chromosome assemblies obtained using this pipeline also allowed recovering piRNA cluster sequences, which was impossible using short-read sequencing. Finally, we used our pipeline to analyze ONT sequencing data from a D. melanogaster unstable line in which LTR transposition was derepressed for 73 successive generations. We could rely on single reads to identify new insertions with intact target site duplications. Moreover, the detailed analysis of TEIs in the wild-type strains and the unstable line did not support the trap model claiming that piRNA clusters are hotspots of TE insertions.
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Wei, Po-Li, Ching-Sheng Hung, Yi-Wei Kao, Ying-Chin Lin, Cheng-Yang Lee, Tzu-Hao Chang, Ben-Chang Shia, and Jung-Chun Lin. "Characterization of Fecal Microbiota with Clinical Specimen Using Long-Read and Short-Read Sequencing Platform." International Journal of Molecular Sciences 21, no. 19 (September 26, 2020): 7110. http://dx.doi.org/10.3390/ijms21197110.
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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.
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Winand, Raf, Bert Bogaerts, Stefan Hoffman, Loïc Lefevre, Maud Delvoye, Julien Van Braekel, Qiang Fu, Nancy HC Roosens, Sigrid CJ De Keersmaecker, and Kevin Vanneste. "Targeting the 16S rRNA Gene for Bacterial Identification in Complex Mixed Samples: Comparative Evaluation of Second (Illumina) and Third (Oxford Nanopore Technologies) Generation Sequencing Technologies." International Journal of Molecular Sciences 21, no. 1 (December 31, 2019): 298. http://dx.doi.org/10.3390/ijms21010298.
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Rapid, accurate bacterial identification in biological samples is an important task for microbiology laboratories, for which 16S rRNA gene Sanger sequencing of cultured isolates is frequently used. In contrast, next-generation sequencing does not require intermediate culturing steps and can be directly applied on communities, but its performance has not been extensively evaluated. We present a comparative evaluation of second (Illumina) and third (Oxford Nanopore Technologies (ONT)) generation sequencing technologies for 16S targeted genomics using a well-characterized reference sample. Different 16S gene regions were amplified and sequenced using the Illumina MiSeq, and analyzed with Mothur. Correct classification was variable, depending on the region amplified. Using a majority vote over all regions, most false positives could be eliminated at the genus level but not the species level. Alternatively, the entire 16S gene was amplified and sequenced using the ONT MinION, and analyzed with Mothur, EPI2ME, and GraphMap. Although >99% of reads were correctly classified at the genus level, up to ≈40% were misclassified at the species level. Both technologies, therefore, allow reliable identification of bacterial genera, but can potentially misguide identification of bacterial species, and constitute viable alternatives to Sanger sequencing for rapid analysis of mixed samples without requiring any culturing steps.
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Massaiu, Ilaria, Paola Songia, Mattia Chiesa, Vincenza Valerio, Donato Moschetta, Valentina Alfieri, Veronika A. Myasoedova, et al. "Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells." International Journal of Molecular Sciences 22, no. 12 (June 12, 2021): 6317. http://dx.doi.org/10.3390/ijms22126317.
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Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-Seq approaches. Oxford Nanopore Technologies (ONT) offers the ability to generate long-read sequencing data in real time via portable protein nanopore USB devices. This work aimed to provide the user with the number of reads that should be sequenced, through the ONT MinION platform, to reach the desired accuracy level for a human cell RNA study. We sequenced three cDNA libraries prepared from poly-adenosine RNA of human primary cardiac fibroblasts. Since the runs were comparable, they were combined in a total dataset of 48 million reads. Synthetic datasets with different sizes were generated starting from the total and analyzed in terms of the number of identified genes and their expression levels. As expected, an improved sensitivity was obtained, increasing the sequencing depth, particularly for the non-coding genes. The reliability of expression levels was assayed by (i) comparison with PCR quantifications of selected genes and (ii) by the implementation of a user-friendly multiplexing method in a single run.
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Lu, Wei, Xinhui Lan, Tong Zhang, Hao Sun, Sanyuan Ma, and Qingyou Xia. "Precise Characterization of Bombyx mori Fibroin Heavy Chain Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies." Insects 12, no. 9 (September 16, 2021): 832. http://dx.doi.org/10.3390/insects12090832.
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To study the evolution of gene function and a species, it is essential to characterize the tandem repetitive sequences distributed across the genome. Cas9-based enrichment combined with nanopore sequencing is an important technique for targeting repetitive sequences. Cpf1 has low molecular weight, low off-target efficiency, and the same editing efficiency as Cas9. There are numerous studies on enrichment sequencing using Cas9 combined with nanopore, while there are only a few studies on the enrichment sequencing of long and highly repetitive genes using Cpf1. We developed Cpf1-based enrichment combined with ONT sequencing (CEO) to characterize the B. mori FibH gene, which is composed of many repeat units with a long and GC-rich sequence up to 17 kb and is not easily amplified by means of a polymerase chain reaction (PCR). CEO has four steps: the dephosphorylation of genomic DNA, the Cpf1 targeted cleavage of FibH, adapter ligation, and ONT sequencing. Using CEO, we determined the fine structure of B. moriFibH, which is 16,845 bp long and includes 12 repetitive domains separated by amorphous regions. Except for the difference of three bases in the intron from the reference gene, the other sequences are identical. Surprisingly, many methylated CG sites were found and distributed unevenly on the FibH repeat unit. The CEO we established is an available means to depict highly repetitive genes, but also a supplement to the enrichment method based on Cas9.
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Batra, Rahul, R. Baldan, P. Cliff, Amita Patel, Jonathan Edgeworth, and M. Chand. "657. Evaluation of Nanopore-Based 16S Ribosomal RNA (rRNA) Gene Sequencing for the Development of a Rapid Infection Intervention Clinical Service." Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S302. http://dx.doi.org/10.1093/ofid/ofz360.725.
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Abstract Background Rapid and accurate identification of bacteria is the basis of appropriate antibiotic treatment and effective clinical decision-making. Next-generation sequencing (NGS) platforms such as Oxford Nanopore Technologies (ONT) holds the promise of a diagnostic revolution by overcoming the limitations of culture-based identification with rapid molecular detection of bacteria. We have developed a pilot to evaluate an ONT 16S rRNA gene assay with the ability to provide real-time analysis and identification of bacterial species. Our aim was to investigate whether long-read sequencing and high-speed analysis can be combined to create a clinically useful, rapid diagnostic tool. Methods A collection of bacterial isolates representing pathogenic species received by the clinical laboratory over 1 year was assembled. Sample preparation was as described in the ONT 16S protocol and included bead beating sample disruption, MagNA Pure automated nucleic acid extraction (Roche), and PCR amplification (Thermo). Sequencing was performed on the MinION and GridION X5 platforms. Output was analyzed with ONT’s automated EPI2ME 16S pipeline which assigns reads to taxa using BLAST results and the NCBI 16S Bacterial database. Results A total of 155 clinical samples with 139 species were sequenced. 119 species were identified at the species level. For 20 samples, a species in the same genus claimed the majority of reads, with the true species being matched to 3%-41% of reads. The average proportion of reads assigned to the correct species was 62.2%, specifically 67% for non-Enterobacteriaceae and 33% for Enterobacteriaceae. 4 clinical samples (3 Bronchoalveolar lavages (BALs), positive for (1) K. pneumoniae, (2) S. pneumoniae, and (3) S. pneumoniae, S. enterica, and S. typhimurium, and 1 bone positive for P. aeruginosa) were also analyzed with sequencing results matching culture. Conclusion Early results show that 16S rRNA sequencing coupled with real-time analysis was able to accelerate pathogen detection and was able to discriminate the majority of species from a relevant clinical collection. Pipeline refinement is required and subsequent confirmatory consensus-based identification may be a helpful adjunct. Nanopore sequencing shows promise as a rapid bacterial pathogen detection platform for clinical service. Disclosures All authors: No reported disclosures.
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Payne, Alexander, Nadine Holmes, Vardhman Rakyan, and Matthew Loose. "BulkVis: a graphical viewer for Oxford nanopore bulk FAST5 files." Bioinformatics 35, no. 13 (November 20, 2018): 2193–98. http://dx.doi.org/10.1093/bioinformatics/bty841.
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Abstract Motivation The Oxford Nanopore Technologies (ONT) MinION is used for sequencing a wide variety of sample types with diverse methods of sample extraction. Nanopore sequencers output FAST5 files containing signal data subsequently base called to FASTQ format. Optionally, ONT devices can collect data from all sequencing channels simultaneously in a bulk FAST5 file enabling inspection of signal in any channel at any point. We sought to visualize this signal to inspect challenging or difficult to sequence samples. Results The BulkVis tool can load a bulk FAST5 file and overlays MinKNOW (the software that controls ONT sequencers) classifications on the signal trace and can show mappings to a reference. Users can navigate to a channel and time or, given a FASTQ header from a read, jump to its specific position. BulkVis can export regions as Nanopore base caller compatible reads. Using BulkVis, we find long reads can be incorrectly divided by MinKNOW resulting in single DNA molecules being split into two or more reads. The longest seen to date is 2 272 580 bases in length and reported in eleven consecutive reads. We provide helper scripts that identify and reconstruct split reads given a sequencing summary file and alignment to a reference. We note that incorrect read splitting appears to vary according to input sample type and is more common in ’ultra-long’ read preparations. Availability and implementation The software is available freely under an MIT license at https://github.com/LooseLab/bulkvis. Supplementary information Supplementary data are available at Bioinformatics online.
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Wang, Shuaibin, Qingwei Song, Shanshan Li, Zhigang Hu, Gangqiang Dong, Chi Song, Hongwen Huang, and Yifei Liu. "Assembly of a Complete Mitogenome of Chrysanthemum nankingense Using Oxford Nanopore Long Reads and the Diversity and Evolution of Asteraceae Mitogenomes." Genes 9, no. 11 (November 12, 2018): 547. http://dx.doi.org/10.3390/genes9110547.
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Diversity in structure and organization is one of the main features of angiosperm mitochondrial genomes (mitogenomes). The ultra-long reads of Oxford Nanopore Technology (ONT) provide an opportunity to obtain a complete mitogenome and investigate the structural variation in unprecedented detail. In this study, we compared mitogenome assembly methods using Illumina and/or ONT sequencing data and obtained the complete mitogenome (208 kb) of Chrysanthemum nankingense based on the hybrid assembly method. The mitogenome encoded 19 transfer RNA genes, three ribosomal RNA genes, and 34 protein-coding genes with 21 group II introns disrupting eight intron-contained genes. A total of seven medium repeats were related to homologous recombination at different frequencies as supported by the long ONT reads. Subsequently, we investigated the variations in gene content and constitution of 28 near-complete mitogenomes from Asteraceae. A total of six protein-coding genes were missing in all Asteraceae mitogenomes, while four other genes were not detected in some lineages. The core fragments (~88 kb) of the Asteraceae mitogenomes had a higher GC content (~46.7%) than the variable and specific fragments. The phylogenetic topology based on the core fragments of the Asteraceae mitogenomes was highly consistent with the topologies obtained from the corresponding plastid datasets. Our results highlighted the advantages of the complete assembly of the C. nankingense mitogenome and the investigation of its structural variation based on ONT sequencing data. Moreover, the method based on local collinear blocks of the mitogenomes could achieve the alignment of highly rearrangeable and variable plant mitogenomes as well as construct a robust phylogenetic topology.
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Chmel, Martin, Oldřich Bartoš, Ondřej Beran, Petr Pajer, Jiří Dresler, Martina Čurdová, and Michal Holub. "Salmonella Paratyphi Infection: Use of Nanopore Sequencing as a Vivid Alternative for the Identification of Invading Bacteria." Prague Medical Report 122, no. 2 (2021): 96–105. http://dx.doi.org/10.14712/23362936.2021.10.
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In our study we present an overview of the use of Oxford Nanopore Technologies (ONT) sequencing technology on the background of Enteric fever. Unlike traditional methods (e.g., qPCR, serological tests), the nanopore sequencing technology enables virtually real-time data generation and highly accurate pathogen identification and characterization. Blood cultures were obtained from a 48-year-old female patient suffering from a high fever, headache and diarrhea. Nevertheless, both the initial serological tests and stool culture appeared to be negative. Therefore, the bacterial isolate from blood culture was used for nanopore sequencing (ONT). This technique in combination with subsequent bioinformatic analyses allowed for prompt identification of the disease-causative agent as Salmonella enterica subsp. enterica serovar Paratyphi A. The National Reference Laboratory for Salmonella (NIPH) independently reported this isolate also as serovar Paratyphi A on the basis of results of biochemical and agglutination tests. Therefore, our results are in concordance with certified standards. Furthermore, the data enabled us to assess some basic questions concerning the comparative genomics, i.e., to describe whether the isolated strain differs from the formerly published ones or not. Quite surprisingly, these results indicate that we have detected a novel and so far, unknown variety of this bacteria.
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Gao, Yan, Bo Liu, Yadong Wang, and Yi Xing. "TideHunter: efficient and sensitive tandem repeat detection from noisy long-reads using seed-and-chain." Bioinformatics 35, no. 14 (July 2019): i200—i207. http://dx.doi.org/10.1093/bioinformatics/btz376.
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Abstract Motivation Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) sequencing technologies can produce long-reads up to tens of kilobases, but with high error rates. In order to reduce sequencing error, Rolling Circle Amplification (RCA) has been used to improve library preparation by amplifying circularized template molecules. Linear products of the RCA contain multiple tandem copies of the template molecule. By integrating additional in silico processing steps, these tandem sequences can be collapsed into a consensus sequence with a higher accuracy than the original raw reads. Existing pipelines using alignment-based methods to discover the tandem repeat patterns from the long-reads are either inefficient or lack sensitivity. Results We present a novel tandem repeat detection and consensus calling tool, TideHunter, to efficiently discover tandem repeat patterns and generate high-quality consensus sequences from amplified tandemly repeated long-read sequencing data. TideHunter works with noisy long-reads (PacBio and ONT) at error rates of up to 20% and does not have any limitation of the maximal repeat pattern size. We benchmarked TideHunter using simulated and real datasets with varying error rates and repeat pattern sizes. TideHunter is tens of times faster than state-of-the-art methods and has a higher sensitivity and accuracy. Availability and implementation TideHunter is written in C, it is open source and is available at https://github.com/yangao07/TideHunter
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Nguyen, Son Hoang, Minh Duc Cao, and Lachlan J. M. Coin. "Real-time resolution of short-read assembly graph using ONT long reads." PLOS Computational Biology 17, no. 1 (January 20, 2021): e1008586. http://dx.doi.org/10.1371/journal.pcbi.1008586.
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A streaming assembly pipeline utilising real-time Oxford Nanopore Technology (ONT) sequencing data is important for saving sequencing resources and reducing time-to-result. A previous approach implemented in npScarf provided an efficient streaming algorithm for hybrid assembly but was relatively prone to mis-assemblies compared to other graph-based methods. Here we present npGraph, a streaming hybrid assembly tool using the assembly graph instead of the separated pre-assembly contigs. It is able to produce more complete genome assembly by resolving the path finding problem on the assembly graph using long reads as the traversing guide. Application to synthetic and real data from bacterial isolate genomes show improved accuracy while still maintaining a low computational cost. npGraph also provides a graphical user interface (GUI) which provides a real-time visualisation of the progress of assembly. The tool and source code is available at https://github.com/hsnguyen/assembly.
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Gigante, Scott. "Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality." F1000Research 6 (March 7, 2017): 227. http://dx.doi.org/10.12688/f1000research.11022.1.
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Oxford Nanopore Technologies' (ONT) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.
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Oliva, Marco, Franco Milicchio, Kaden King, Grace Benson, Christina Boucher, and Mattia Prosperi. "Portable nanopore analytics: are we there yet?" Bioinformatics 36, no. 16 (April 11, 2020): 4399–405. http://dx.doi.org/10.1093/bioinformatics/btaa237.
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Abstract Motivation Oxford Nanopore technologies (ONT) add miniaturization and real time to high-throughput sequencing. All available software for ONT data analytics run on cloud/clusters or personal computers. Instead, a linchpin to true portability is software that works on mobile devices of internet connections. Smartphones’ and tablets’ chipset/memory/operating systems differ from desktop computers, but software can be recompiled. We sought to understand how portable current ONT analysis methods are. Results Several tools, from base-calling to genome assembly, were ported and benchmarked on an Android smartphone. Out of 23 programs, 11 succeeded. Recompilation failures included lack of standard headers and unsupported instruction sets. Only DSK, BCALM2 and Kraken were able to process files up to 16 GB, with linearly scaling CPU-times. However, peak CPU temperatures were high. In conclusion, the portability scenario is not favorable. Given the fast market growth, attention of developers to ARM chipsets and Android/iOS is warranted, as well as initiatives to implement mobile-specific libraries. Availability and implementation The source code is freely available at: https://github.com/marco-oliva/portable-nanopore-analytics.
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Hansen, Katrine Hartung, Minna Rud Andreasen, Martin Schou Pedersen, Henrik Westh, Lotte Jelsbak, and Kristian Schønning. "Resistance to piperacillin/tazobactam in Escherichia coli resulting from extensive IS26-associated gene amplification of blaTEM-1." Journal of Antimicrobial Chemotherapy 74, no. 11 (August 14, 2019): 3179–83. http://dx.doi.org/10.1093/jac/dkz349.
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Abstract Background bla TEM-1 encodes a narrow-spectrum β-lactamase that is inhibited by β-lactamase inhibitors and commonly present in Escherichia coli. Hyperproduction of blaTEM-1 may cause resistance to penicillin/β-lactamase inhibitor (P/BLI) combinations. Objectives To characterize EC78, an E. coli bloodstream isolate, resistant to P/BLI combinations, which contains extensive amplification of blaTEM-1 within the chromosome. Methods EC78 was sequenced using Illumina and Oxford Nanopore Technology (ONT) methodology. Configuration of blaTEM-1 amplification was probed using PCR. Expression of blaTEM-1 mRNA was determined using quantitative PCR and β-lactamase activity was determined spectrophotometrically in a nitrocefin conversion assay. Growth rate was assessed to determine fitness and stability of the gene amplification was assessed by passage in the absence of antibiotics. Results Illumina sequencing of EC78 identified blaTEM-1B as the only acquired β-lactamase preceded by the WT P3 promoter and present at a copy number of 182.6 with blaTEM-1B bracketed by IS26 elements. The chromosomal location of the IS26-blaTEM-1B amplification was confirmed by ONT sequencing. Hyperproduction of blaTEM-1 was confirmed by increased transcription of blaTEM-1 and β-lactamase activity and associated with a significant fitness cost; however, the array was maintained at a relatively high copy number for 150 generations. PCR screening for blaTEM amplification of isolates resistant to P/BLI combinations identified an additional strain containing an IS26-associated amplification of a blaTEM gene. Conclusions IS26-associated amplification of blaTEM can cause resistance to P/BLI combinations. This adaptive mechanism of resistance may be overlooked if simple methods of genotypic prediction (e.g. gene presence/absence) are used to predict antimicrobial susceptibility from sequencing data.
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Ip, Camilla L. C., Matthew Loose, John R. Tyson, Mariateresa de Cesare, Bonnie L. Brown, Miten Jain, Richard M. Leggett, et al. "MinION Analysis and Reference Consortium: Phase 1 data release and analysis." F1000Research 4 (October 15, 2015): 1075. http://dx.doi.org/10.12688/f1000research.7201.1.
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The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance.
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Du, Chenghao. "The Power of Using Novel Nanopore Sequencing Technology for Diagnosis, Genomic and Pathological Studies of Covid-19." South Florida Journal of Development 2, no. 3 (July 8, 2021): 4014–28. http://dx.doi.org/10.46932/sfjdv2n3-017.
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The novel coronavirus disease 2019 (COVID‐19), originally identified in December 2019 Wuhan, China, has propagated to worldwide pandemic, causing many cases of death and morbidity. Since the development of COVID-19 vaccines is still under experimental stages without public access, different types of testing and detection ensuring rapid and accurate results are urgently required to prevent delaying isolation of infected patients. The traditional diagnostic and analytical methods of COVID-19 relied heavily on nucleic acid and antibody-antigen methods but are subject to assembly bias, restricted by reading length, showed some false positive/negative results and had a long turnaround time. Hence, three styles of nanopore sequencing techniques as complementary tools for COVID-19 diagnosis and analysis are introduced. The long-read nanopore sequencing technology has been adopted in metagenomic and pathological studies of virosphere including SARS-CoV-2 recently by either metagenomically, directly or indirectly sequencing the viral genomic RNA of SARS-CoV-2 in real-time to detect infected specimens for early isolation and treatment, to investigate the transmission and evolutionary routes of SARS-CoV-2 as well as its pathogenicity and epidemiology. In this article, the Nanopore-Based Metagenomic Sequencing, Direct RNA Nanopore Sequencing (DRS), and Nanopore Targeted Sequencing (NTS) become the main focus of the novel COVID-19 detecting analytical methods in sequencing platforms, which are discussed in comparison with other traditional and popular diagnostic methods. Finally, different types of nanopore sequencing platforms that are developed by Oxford Nanopore Technologies (ONT) due to various purposes and demands in viral genomic research are briefly discussed.
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Du, Chenghao. "The Power of Using Novel Nanopore Sequencing Technology for Diagnosis, Genomic and Pathological Studies of Covid-19." E3S Web of Conferences 271 (2021): 04024. http://dx.doi.org/10.1051/e3sconf/202127104024.
Full textAbstract:
The novel coronavirus disease 2019 (COVID‐19), originally identified in December 2019 Wuhan, China, has propagated to worldwide pandemic, causing many cases of death and morbidity. Since the development of COVID-19 vaccines is still under experimental stages without public access, different types of testing and detection ensuring rapid and accurate results are urgently required to prevent delaying isolation of infected patients. The traditional diagnostic and analytical methods of COVID-19 relied heavily on nucleic acid and antibody-antigen methods but are subject to assembly bias, restricted by reading length, showed some false positive/negative results and had a long turnaround time. Hence, three styles of nanopore sequencing techniques as complementary tools for COVID-19 diagnosis and analysis are introduced. The long-read nanopore sequencing technology has been adopted in metagenomic and pathological studies of virosphere including SARS-CoV-2 recently by either metagenomically, directly or indirectly sequencing the viral genomic RNA of SARS-CoV-2 in real-time to detect infected specimens for early isolation and treatment, to investigate the transmission and evolutionary routes of SARS-CoV-2 as well as its pathogenicity and epidemiology. In this article, the Nanopore-Based Metagenomic Sequencing, Direct RNA Nanopore Sequencing (DRS), and Nanopore Targeted Sequencing (NTS) become the main focus of the novel COVID-19 detecting analytical methods in sequencing platforms, which are discussed in comparison with other traditional and popular diagnostic methods. Finally, different types of nanopore sequencing platforms that are developed by Oxford Nanopore Technologies (ONT) due to various purposes and demands in viral genomic research are briefly discussed.
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Lee, Yun Gyeong, Sang Chul Choi, Yuna Kang, Kyeong Min Kim, Chon-Sik Kang, and Changsoo Kim. "Constructing a Reference Genome in a Single Lab: The Possibility to Use Oxford Nanopore Technology." Plants 8, no. 8 (August 6, 2019): 270. http://dx.doi.org/10.3390/plants8080270.
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The whole genome sequencing (WGS) has become a crucial tool in understanding genome structure and genetic variation. The MinION sequencing of Oxford Nanopore Technologies (ONT) is an excellent approach for performing WGS and it has advantages in comparison with other Next-Generation Sequencing (NGS): It is relatively inexpensive, portable, has simple library preparation, can be monitored in real-time, and has no theoretical limits on reading length. Sorghum bicolor (L.) Moench is diploid (2n = 2x = 20) with a genome size of about 730 Mb, and its genome sequence information is released in the Phytozome database. Therefore, sorghum can be used as a good reference. However, plant species have complex and large genomes when compared to animals or microorganisms. As a result, complete genome sequencing is difficult for plant species. MinION sequencing that produces long-reads can be an excellent tool for overcoming the weak assembly of short-reads generated from NGS by minimizing the generation of gaps or covering the repetitive sequence that appears on the plant genome. Here, we conducted the genome sequencing for S. bicolor cv. BTx623 while using the MinION platform and obtained 895,678 reads and 17.9 gigabytes (Gb) (ca. 25× coverage of reference) from long-read sequence data. A total of 6124 contigs (covering 45.9%) were generated from Canu, and a total of 2661 contigs (covering 50%) were generated from Minimap and Miniasm with a Racon through a de novo assembly using two different tools and mapped assembled contigs against the sorghum reference genome. Our results provide an optimal series of long-read sequencing analysis for plant species while using the MinION platform and a clue to determine the total sequencing scale for optimal coverage that is based on various genome sizes.
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Nguyen, Scott V., David R. Greig, Daniel Hurley, Orla Donoghue, Yu Cao, Evonne McCabe, Molly Mitchell, Kirsten Schaffer, Claire Jenkins, and Séamus Fanning. "Yersinia canariae sp. nov., isolated from a human yersiniosis case." International Journal of Systematic and Evolutionary Microbiology 70, no. 4 (April 1, 2020): 2382–87. http://dx.doi.org/10.1099/ijsem.0.004047.
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A Gram-negative rod from the Yersinia genus was isolated from a clinical case of yersiniosis in the United Kingdom. Long read sequencing data from an Oxford Nanopore Technologies (ONT) MinION in conjunction with Illumina HiSeq reads were used to generate a finished quality genome of this strain. Overall Genome Related Index (OGRI) of the strain was used to determine that it was a novel species within Yersinia , despite biochemical similarities to Yersinia enterocolitica . The 16S ribosomal RNA gene accessions are MN434982-MN434987 and the accession number for the complete and closed chromosome is CP043727. The type strain is SRR7544370T (=NCTC 14382T/=LMG 31573T).
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Ren, Jingwen, and Mark J. P. Chaisson. "lra: A long read aligner for sequences and contigs." PLOS Computational Biology 17, no. 6 (June 21, 2021): e1009078. http://dx.doi.org/10.1371/journal.pcbi.1009078.
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It is computationally challenging to detect variation by aligning single-molecule sequencing (SMS) reads, or contigs from SMS assemblies. One approach to efficiently align SMS reads is sparse dynamic programming (SDP), where optimal chains of exact matches are found between the sequence and the genome. While straightforward implementations of SDP penalize gaps with a cost that is a linear function of gap length, biological variation is more accurately represented when gap cost is a concave function of gap length. We have developed a method, lra, that uses SDP with a concave-cost gap penalty, and used lra to align long-read sequences from PacBio and Oxford Nanopore (ONT) instruments as well as de novo assembly contigs. This alignment approach increases sensitivity and specificity for SV discovery, particularly for variants above 1kb and when discovering variation from ONT reads, while having runtime that are comparable (1.05-3.76×) to current methods. When applied to calling variation from de novo assembly contigs, there is a 3.2% increase in Truvari F1 score compared to minimap2+htsbox. lra is available in bioconda (https://anaconda.org/bioconda/lra) and github (https://github.com/ChaissonLab/LRA).
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Mechan Llontop, Marco E., Parul Sharma, Marcela Aguilera Flores, Shu Yang, Jill Pollok, Long Tian, Chenjie Huang, et al. "Strain-Level Identification of Bacterial Tomato Pathogens Directly from Metagenomic Sequences." Phytopathology® 110, no. 4 (April 2020): 768–79. http://dx.doi.org/10.1094/phyto-09-19-0351-r.
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Routine strain-level identification of plant pathogens directly from symptomatic tissue could significantly improve plant disease control and prevention. Here we tested the Oxford Nanopore Technologies (ONT) MinION sequencer for metagenomic sequencing of tomato plants either artificially inoculated with a known strain of the bacterial speck pathogen Pseudomonas syringae pv. tomato or collected in the field and showing bacterial spot symptoms caused by one of four Xanthomonas species. After species-level identification via ONT’s WIMP software and the third-party tools Sourmash and MetaMaps, we used Sourmash and MetaMaps with a custom database of representative genomes of bacterial tomato pathogens to attempt strain-level identification. In parallel, each metagenome was assembled and the longest contigs were used as query with the genome-based microbial identification Web service LINbase. Both the read-based and assembly-based approaches correctly identified P. syringae pv. tomato strain T1 in the artificially inoculated samples. The pathogen strain in most field samples was identified as a member of Xanthomonas perforans group 2. This result was confirmed by whole genome sequencing of colonies isolated from one of the samples. Although in our case metagenome-based pathogen identification at the strain level was achieved, caution still must be exercised in interpreting strain-level results because of the challenges inherent to assigning reads to specific strains and the error rate of nanopore sequencing.
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Juraschek, Katharina, Maria Borowiak, Simon H. Tausch, Burkhard Malorny, Annemarie Käsbohrer, Saria Otani, Stefan Schwarz, Diana Meemken, Carlus Deneke, and Jens Andre Hammerl. "Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli." Microorganisms 9, no. 3 (March 14, 2021): 598. http://dx.doi.org/10.3390/microorganisms9030598.
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Antimicrobial resistance (AMR) is a major threat to public health worldwide. Currently, AMR typing changes from phenotypic testing to whole-genome sequence (WGS)-based detection of resistance determinants for a better understanding of the isolate diversity and elements involved in gene transmission (e.g., plasmids, bacteriophages, transposons). However, the use of WGS data in monitoring purposes requires suitable techniques, standardized parameters and approved guidelines for reliable AMR gene detection and prediction of their association with mobile genetic elements (plasmids). In this study, different sequencing and assembly strategies were tested for their suitability in AMR monitoring in Escherichia coli in the routines of the German National Reference Laboratory for Antimicrobial Resistances. To assess the outcomes of the different approaches, results from in silico predictions were compared with conventional phenotypic- and genotypic-typing data. With the focus on (fluoro)quinolone-resistant E.coli, five qnrS-positive isolates with multiple extrachromosomal elements were subjected to WGS with NextSeq (Illumina), PacBio (Pacific BioSciences) and ONT (Oxford Nanopore) for in depth characterization of the qnrS1-carrying plasmids. Raw reads from short- and long-read sequencing were assembled individually by Unicycler or Flye or a combination of both (hybrid assembly). The generated contigs were subjected to bioinformatics analysis. Based on the generated data, assembly of long-read sequences are error prone and can yield in a loss of small plasmid genomes. In contrast, short-read sequencing was shown to be insufficient for the prediction of a linkage of AMR genes (e.g., qnrS1) to specific plasmid sequences. Furthermore, short-read sequencing failed to detect certain duplications and was unsuitable for genome finishing. Overall, the hybrid assembly led to the most comprehensive typing results, especially in predicting associations of AMR genes and mobile genetic elements. Thus, the use of different sequencing technologies and hybrid assemblies currently represents the best approach for reliable AMR typing and risk assessment.
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Sun, Jin, Runsheng Li, Chong Chen, Julia D. Sigwart, and Kevin M. Kocot. "Benchmarking Oxford Nanopore read assemblers for high-quality molluscan genomes." Philosophical Transactions of the Royal Society B: Biological Sciences 376, no. 1825 (April 5, 2021): 20200160. http://dx.doi.org/10.1098/rstb.2020.0160.
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Choosing the optimum assembly approach is essential to achieving a high-quality genome assembly suitable for comparative and evolutionary genomic investigations. Significant recent progress in long-read sequencing technologies such as PacBio and Oxford Nanopore Technologies (ONT) has also brought about a large variety of assemblers. Although these have been extensively tested on model species such as Homo sapiens and Drosophila melanogaster , such benchmarking has not been done in Mollusca, which lacks widely adopted model species. Molluscan genomes are notoriously rich in repeats and are often highly heterozygous, making their assembly challenging. Here, we benchmarked 10 assemblers based on ONT raw reads from two published molluscan genomes of differing properties, the gastropod Chrysomallon squamiferum (356.6 Mb, 1.59% heterozygosity) and the bivalve Mytilus coruscus (1593 Mb, 1.94% heterozygosity). By optimizing the assembly pipeline, we greatly improved both genomes from previously published versions. Our results suggested that 40–50X of ONT reads are sufficient for high-quality genomes, with Flye being the recommended assembler for compact and less heterozygous genomes exemplified by C. squamiferum , while NextDenovo excelled for more repetitive and heterozygous molluscan genomes exemplified by M. coruscus . A phylogenomic analysis using the two updated genomes with 32 other published high-quality lophotrochozoan genomes resulted in maximum support across all nodes, and we show that improved genome quality also leads to more complete matrices for phylogenomic inferences. Our benchmarking will ensure efficiency in future assemblies for molluscs and perhaps also for other marine phyla with few genomes available. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.
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Waweru, Jacqueline Wahura, Zaydah de Laurent, Everlyn Kamau, Khadija Said, Elijah Gicheru, Martin Mutunga, Caleb Kibet, et al. "Enrichment approach for unbiased sequencing of respiratory syncytial virus directly from clinical samples." Wellcome Open Research 6 (May 7, 2021): 99. http://dx.doi.org/10.12688/wellcomeopenres.16756.1.
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Background: Nasopharyngeal samples contain higher quantities of bacterial and host nucleic acids relative to viruses; presenting challenges during virus metagenomics sequencing, which underpins agnostic sequencing protocols. We aimed to develop a viral enrichment protocol for unbiased whole-genome sequencing of respiratory syncytial virus (RSV) from nasopharyngeal samples using the Oxford Nanopore Technology (ONT) MinION platform. Methods: We assessed two protocols using RSV positive samples. Protocol 1 involved physical pre-treatment of samples by centrifugal processing before RNA extraction, while Protocol 2 entailed direct RNA extraction without prior enrichment. Concentrates from Protocol 1 and RNA extracts from Protocol 2 were each divided into two fractions; one was DNase treated while the other was not. RNA was then extracted from both concentrate fractions per sample and RNA from both protocols converted to cDNA, which was then amplified using the tagged Endoh primers through Sequence-Independent Single-Primer Amplification (SISPA) approach, a library prepared, and sequencing done. Statistical significance during analysis was tested using the Wilcoxon signed-rank test. Results: DNase-treated fractions from both protocols recorded significantly reduced host and bacterial contamination unlike the untreated fractions (in each protocol p<0.01). Additionally, DNase treatment after RNA extraction (Protocol 2) enhanced host and bacterial read reduction compared to when done before (Protocol 1). However, neither protocol yielded whole RSV genomes. Sequenced reads mapped to parts of the nucleoprotein (N gene) and polymerase complex (L gene) from Protocol 1 and 2, respectively. Conclusions: DNase treatment was most effective in reducing host and bacterial contamination, but its effectiveness improved if done after RNA extraction than before. We attribute the incomplete genome segments to amplification biases resulting from the use of short length random sequence (6 bases) in tagged Endoh primers. Increasing the length of the random nucleotides from six hexamers to nine or 12 in future studies may reduce the coverage biases.
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Kot, Witold, Nikoline S. Olsen, Tue K. Nielsen, Geoffrey Hutinet, Valérie de Crécy-Lagard, Liang Cui, Peter C. Dedon, et al. "Detection of preQ0 deazaguanine modifications in bacteriophage CAjan DNA using Nanopore sequencing reveals same hypermodification at two distinct DNA motifs." Nucleic Acids Research 48, no. 18 (September 17, 2020): 10383–96. http://dx.doi.org/10.1093/nar/gkaa735.
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Abstract In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.
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Klever, Marius-Konstantin, Eric Sträng, Julius Jungnitsch, Uirá Souto Melo, Sara Hetzel, Anna Dolnik, Robert Schöpflin, et al. "Integration of Hi-C and Nanopore Sequencing for Structural Variant Analysis in AML with a Complex Karyotype: (Chromothripsis)²." Blood 136, Supplement 1 (November 5, 2020): 28. http://dx.doi.org/10.1182/blood-2020-133787.
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Background. Acute myeloid leukemia (AML) with a complex karyotype (CK-AML) is an AML subtype with a still dismal outcome despite recent therapeutic advances. The prognosis is even worse when the underlying structural variants (SVs) lead to an extremely complex pattern of rearrangements, called chromothripsis, with a median overall survival of only 120 days. Except for the presence of inactivating TP53 aberrations in about 70% of all AML-CK cases, the pathogenesis is poorly understood. To gain novel insights into the molecular mechanisms underlying CK-AML reliable high precision SV delineation is needed, which so far has been a major limitation in cancer research. Aim. We developed a SV detection pipeline by integrating Oxford Nanopore Technology (ONT) based whole genome sequencing (WGS) and Hi-C sequencing. This pipeline generated precise characterization of SVs for which the impact on gene expression and the emergence of novel fusion genes was studied by RNA-seq and ONT transcriptome sequencing. Patients and Methods. We applied our WGS and Hi-C SV detection pipeline to a cohort of 11 AML-CK cases. Nanopore DNA Sequencing was performed until a genomic coverage >10x per patient was reached. The samples of 9 patients were also subjected to Nanopore cDNA sequencing for fusion gene analysis and Illumina based RNA-seq for transcript quantification. As controls for Hi-C and Illumina RNA sequencing, CD34+ hematopoietic stem cell enriched samples from five healthy donors were used. Results. Our SV detection pipeline enabled us to fully reconstruct the derivate chromosome structure even of very complex, chromothriptic rearrangements in CK-AML. This enabled us to identify features of chromothripsis, that could previously not be detected using conventual technologies. We found local clustering of breakpoints in three of the patients with up to 31 Inversions and Translocations located in a genomic region of just 2.7 kb. These breakpoints were present in the Hi-C as well as in our Nanopore SV dataset. Our SV pipeline also showed that in these highly clustered regions, the very small rejoined fragments (in many cases less than 1 kb in size) often showed an elevated copy number (CN) state, i.e. small amplifications. We termed this newly discovered phenomenon chromothripsis-in-chromothripsis or (chromothripsis)². The precise knowledge about these breakpoints, which were validated by two different technologies, enabled us to study the pathogenesis of CK-AML at a so far unprecedented resolution. Fusion transcripts could be very precisely mapped and the impact of the breakpoints and CN changes on gene expression levels could be validated, thereby indicating functional relevance of the respective aberrations. Conclusions. The combination of Hi-C and long-read sequencing for SV detection proved to be a powerful tool for precise SV detection. Our SV pipeline allowed us to discover a new level of complexity in chromothripsis. Application of this pipeline to leukemias as well as other types of cancer can improve the precision of SV detection, thereby raising new opportunities for functional interpretation of complex genomic aberrations of pathogenic relevance. Disclosures Döhner: Sunesis Pharmaceuticals: Research Funding; Astex Pharmaceuticals: Consultancy; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Arog: Research Funding; Roche: Consultancy; Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Abbvie: Consultancy; Agios: Consultancy; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Astellas Pharma: Consultancy; Celgene: Consultancy, Honoraria. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Bullinger:Amgen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees.
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Kubiak-Szymendera, Monika, Leszek P. Pryszcz, Wojciech Białas, and Ewelina Celińska. "Epigenetic Response of Yarrowia lipolytica to Stress: Tracking Methylation Level and Search for Methylation Patterns via Whole-Genome Sequencing." Microorganisms 9, no. 9 (August 24, 2021): 1798. http://dx.doi.org/10.3390/microorganisms9091798.
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DNA methylation is a common, but not universal, epigenetic modification that plays an important role in multiple cellular processes. While definitely settled for numerous plant, mammalian, and bacterial species, the genome methylation in different fungal species, including widely studied and industrially-relevant yeast species, Yarrowia lipolytica, is still a matter of debate. In this paper, we report a differential DNA methylation level in the genome of Y. lipolytica subjected to sequential subculturing and to heat stress conditions. To this end, we adopted repeated batch bioreactor cultivations of Y. lipolytica subjected to thermal stress in specific time intervals. To analyze the variation in DNA methylation between stressed and control cultures, we (a) quantified the global DNA methylation status using an immuno-assay, and (b) studied DNA methylation patterns through whole-genome sequencing. Primarily, we demonstrated that 5 mC modification can be detected using a commercial immuno-assay, and that the modifications are present in Y. lipolytica’s genome at ~0.5% 5 mC frequency. On the other hand, we did not observe any changes in the epigenetic response of Y. lipolytica to heat shock (HS) treatment. Interestingly, we identified a general phenomenon of decreased 5 mC level in Y. lipolytica’s genome in the stationary phase of growth, when compared to a late-exponential epigenome. While this study provides an insight into the subculturing stress response and adaptation to the stress at epigenetic level by Y. lipolytica, it also leaves an open question of inability to detect any genomic DNA methylation level (either in CpG context or context-less) through whole-genome sequencing. The results of ONT sequencing, suggesting that 5 mC modification is either rare or non-existent in Y. lipolytica genome, are contradicted with the results of the immunoassay.
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Dippenaar, Anzaan, Sander N. Goossens, Melanie Grobbelaar, Selien Oostvogels, Bart Cuypers, Kris Laukens, Conor J. Meehan, Robin M. Warren, and Annelies van Rie. "Nanopore sequencing for Mycobacterium tuberculosis : a critical review of the literature, new developments and future opportunities." Journal of Clinical Microbiology, June 16, 2021. http://dx.doi.org/10.1128/jcm.00646-21.
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The next-generation short-read sequencing technologies that generate comprehensive, whole-genome data with single-nucleotide resolution have already advanced tuberculosis diagnosis, treatment, surveillance and source investigation. Their high costs, tedious and lengthy processes, and large equipment remain major hurdles for research use in high tuberculosis burden countries and implementation into routine care. The portable next-generation sequencing devices developed by Oxford Nanopore Technologies (ONT) are attractive alternatives due to their long-read sequence capability, compact low-cost hardware, and continued improvements in accuracy and throughput. A systematic review of the published literature demonstrated limited uptake of ONT sequencing in tuberculosis research and clinical care. Of the 12 eligible articles presenting ONT sequencing data on at least one Mycobacterium tuberculosis sample, four addressed software development for long read ONT sequencing data with potential applications for M. tuberculosis . Only eight studies presented results of ONT sequencing of M. tuberculosis , of which five performed whole-genome and three did targeted sequencing. Based on these findings, we summarize the standard processes, reflect on the current limitations of ONT sequencing technology, and the research needed to overcome the main hurdles. Summary: The low capital cost, portable nature and continued improvement in the performance of ONT sequencing make it an attractive option for sequencing for research and clinical care, but limited data is available on its application in the tuberculosis field. Important research investment is needed to unleash the full potential of ONT sequencing for tuberculosis research and care.