2025
Meyer, Daria; Hennig, Anne; Hums, Anna-Bawany; Guntinas-Lichius, Orlando; Schmitz, Martina; Marz, Manja
Nanopore sequencing-derived methylation biomarker prediction for methylation-specific PCR in patients with head and neck squamous cell carcinoma Journal Article
In: Clinical Epigenetics, vol. 17, 2025.
Abstract | Links | BibTeX | Tags: cancer, nanopore, nucleic acid modifications
@article{nokey_88,
title = {Nanopore sequencing-derived methylation biomarker prediction for methylation-specific PCR in patients with head and neck squamous cell carcinoma},
author = {Daria Meyer and Anne Hennig and Anna-Bawany Hums and Orlando Guntinas-Lichius and Martina Schmitz and Manja Marz },
doi = {10.1186/s13148-025-01960-7},
year = {2025},
date = {2025-09-13},
journal = {Clinical Epigenetics},
volume = {17},
abstract = {DNA methylation of CpG islands is altered in cancer cells. Hypermethylation of single CpG islands in the promoter regions of tumor-suppressor genes occurs already in the early stages of cancer. These methylation changes are cancer-type specific and therefore can serve as early cancer biomarker. Identifying good and reliable biomarkers is crucial for the development of diagnostic tests and their application in clinical practice and remains the most significant challenge to date.},
keywords = {cancer, nanopore, nucleic acid modifications},
pubstate = {published},
tppubtype = {article}
}
DNA methylation of CpG islands is altered in cancer cells. Hypermethylation of single CpG islands in the promoter regions of tumor-suppressor genes occurs already in the early stages of cancer. These methylation changes are cancer-type specific and therefore can serve as early cancer biomarker. Identifying good and reliable biomarkers is crucial for the development of diagnostic tests and their application in clinical practice and remains the most significant challenge to date.
Thomas, Christine; Brangsch, Hanka; Galeone, Valentina; Hölzer, Martin; Marz, Manja; Linde, Jörg
Accurately assembling nanopore sequencing data of highly pathogenic bacteria Journal Article
In: BMC Genomics, vol. 26, 2025.
Abstract | Links | BibTeX | Tags: assembly, bacteria, DNA / genomics, nanopore
@article{nokey_82,
title = {Accurately assembling nanopore sequencing data of highly pathogenic bacteria},
author = {Christine Thomas and Hanka Brangsch and Valentina Galeone and Martin Hölzer and Manja Marz and Jörg Linde},
doi = {10.1186/s12864-025-11793-6},
year = {2025},
date = {2025-08-28},
journal = {BMC Genomics},
volume = {26},
abstract = {Background: Bacterial genome exploration and outbreak analysis rely heavily on robust whole-genome sequencing and bioinformatics analysis. Widely-used genomic methods, such as genotyping and detection of genetic markers demand high sequencing accuracy and precise genome assembly for reliable results.
Methods: To assess the utility of nanopore sequencing for genotyping highly pathogenic bacteria with low mutation rates, we sequenced six reference strains using Oxford Nanopore Technologies (ONT) R10.4.1 chemistry and Illumina and evaluated different assembly strategies. The publicly available RefSeq assemblies were chosen as the ground truth. Publicly available sequencing data from key foodborne and public-health-related bacterial pathogens were examined to provide a broader context for the analysis.
Results: While for Bacillus (Ba.) anthracis an almost perfect assembly was achieved, results varied for other species. For Brucella (Br.) spp., the final assemblies comprised five to 46 different nucleotides in comparison to Sanger-sequenced references. For some key foodborne and public-health-related bacterial pathogens (Klebsiella (K.) variicola, Listeria spp., Mycobacterium (M.) tuberculosis, Staphylococcus (Sta.) aureus, and Streptococcus (Str.) pyogenes) perfect genomes were obtained. Enhanced basecalling models have generally improved assembly accuracy, however, for certain species such as Br. abortus, older models have produced higher accuracy. While long-read polishing mainly improves assembly quality with only one round needed, our results indicate that this process may also degrade assembly quality. Overall, 81% of the observed errors in ONT assemblies were located within coding sequences (CDS). Furthermore, we found that methylation caused 6.5% of the errors, and the bacterial methylation-aware medaka polishing model reduced the number of errors linked to methylation. Core-genome Multilocus Sequence Typing (cgMLST) analysis revealed allele differences in Ba. anthracis, Br. abortus, and Francisella (F.) tularensis for some assemblers, although with fewer than five allele differences. In the case of Br. melitensis, some assemblies included five allele differences, whereas for Br. suis the correct cgMLST alleles were observed.
Conclusions: Assembling nanopore data from pathogenic bacteria vary in quality across different species and methods. However, errors persist in the final assemblies, including within cgMLST loci, influencing the reliability of outbreak predictions. Nevertheless, specific combinations of existing tools can generate perfect genome assemblies from bacterial ONT sequencing data for outbreak analysis without short-read polishing.},
keywords = {assembly, bacteria, DNA / genomics, nanopore},
pubstate = {published},
tppubtype = {article}
}
Background: Bacterial genome exploration and outbreak analysis rely heavily on robust whole-genome sequencing and bioinformatics analysis. Widely-used genomic methods, such as genotyping and detection of genetic markers demand high sequencing accuracy and precise genome assembly for reliable results.
Methods: To assess the utility of nanopore sequencing for genotyping highly pathogenic bacteria with low mutation rates, we sequenced six reference strains using Oxford Nanopore Technologies (ONT) R10.4.1 chemistry and Illumina and evaluated different assembly strategies. The publicly available RefSeq assemblies were chosen as the ground truth. Publicly available sequencing data from key foodborne and public-health-related bacterial pathogens were examined to provide a broader context for the analysis.
Results: While for Bacillus (Ba.) anthracis an almost perfect assembly was achieved, results varied for other species. For Brucella (Br.) spp., the final assemblies comprised five to 46 different nucleotides in comparison to Sanger-sequenced references. For some key foodborne and public-health-related bacterial pathogens (Klebsiella (K.) variicola, Listeria spp., Mycobacterium (M.) tuberculosis, Staphylococcus (Sta.) aureus, and Streptococcus (Str.) pyogenes) perfect genomes were obtained. Enhanced basecalling models have generally improved assembly accuracy, however, for certain species such as Br. abortus, older models have produced higher accuracy. While long-read polishing mainly improves assembly quality with only one round needed, our results indicate that this process may also degrade assembly quality. Overall, 81% of the observed errors in ONT assemblies were located within coding sequences (CDS). Furthermore, we found that methylation caused 6.5% of the errors, and the bacterial methylation-aware medaka polishing model reduced the number of errors linked to methylation. Core-genome Multilocus Sequence Typing (cgMLST) analysis revealed allele differences in Ba. anthracis, Br. abortus, and Francisella (F.) tularensis for some assemblers, although with fewer than five allele differences. In the case of Br. melitensis, some assemblies included five allele differences, whereas for Br. suis the correct cgMLST alleles were observed.
Conclusions: Assembling nanopore data from pathogenic bacteria vary in quality across different species and methods. However, errors persist in the final assemblies, including within cgMLST loci, influencing the reliability of outbreak predictions. Nevertheless, specific combinations of existing tools can generate perfect genome assemblies from bacterial ONT sequencing data for outbreak analysis without short-read polishing.
Methods: To assess the utility of nanopore sequencing for genotyping highly pathogenic bacteria with low mutation rates, we sequenced six reference strains using Oxford Nanopore Technologies (ONT) R10.4.1 chemistry and Illumina and evaluated different assembly strategies. The publicly available RefSeq assemblies were chosen as the ground truth. Publicly available sequencing data from key foodborne and public-health-related bacterial pathogens were examined to provide a broader context for the analysis.
Results: While for Bacillus (Ba.) anthracis an almost perfect assembly was achieved, results varied for other species. For Brucella (Br.) spp., the final assemblies comprised five to 46 different nucleotides in comparison to Sanger-sequenced references. For some key foodborne and public-health-related bacterial pathogens (Klebsiella (K.) variicola, Listeria spp., Mycobacterium (M.) tuberculosis, Staphylococcus (Sta.) aureus, and Streptococcus (Str.) pyogenes) perfect genomes were obtained. Enhanced basecalling models have generally improved assembly accuracy, however, for certain species such as Br. abortus, older models have produced higher accuracy. While long-read polishing mainly improves assembly quality with only one round needed, our results indicate that this process may also degrade assembly quality. Overall, 81% of the observed errors in ONT assemblies were located within coding sequences (CDS). Furthermore, we found that methylation caused 6.5% of the errors, and the bacterial methylation-aware medaka polishing model reduced the number of errors linked to methylation. Core-genome Multilocus Sequence Typing (cgMLST) analysis revealed allele differences in Ba. anthracis, Br. abortus, and Francisella (F.) tularensis for some assemblers, although with fewer than five allele differences. In the case of Br. melitensis, some assemblies included five allele differences, whereas for Br. suis the correct cgMLST alleles were observed.
Conclusions: Assembling nanopore data from pathogenic bacteria vary in quality across different species and methods. However, errors persist in the final assemblies, including within cgMLST loci, influencing the reliability of outbreak predictions. Nevertheless, specific combinations of existing tools can generate perfect genome assemblies from bacterial ONT sequencing data for outbreak analysis without short-read polishing.
Lataretu, Marie; Krautwurst, Sebastian; Huska, Matthew R; Marquet, Mike; Viehweger, Adrian; Braun, Sascha D; Brandt, Christian; Hölzer, Martin
Targeted decontamination of sequencing data with CLEAN Journal Article
In: NAR Genomics and Bioinformatics, vol. 7, 2025.
Abstract | Links | BibTeX | Tags: assembly, metagenomics, nanopore, RNA / transcriptomics, software
@article{nokey_81,
title = {Targeted decontamination of sequencing data with CLEAN},
author = {Marie Lataretu and Sebastian Krautwurst and Matthew R Huska and Mike Marquet and Adrian Viehweger and Sascha D Braun and Christian Brandt and Martin Hölzer},
doi = {10.1093/nargab/lqaf105},
year = {2025},
date = {2025-07-04},
urldate = {2025-07-04},
journal = {NAR Genomics and Bioinformatics},
volume = {7},
abstract = {Many biological and medical questions are answered based on the analysis of sequence data. However, we can find contamination, artificial spike-ins, and overrepresented rRNA (ribosomal RNA) sequences in various read collections and assemblies. In particular, spike-ins used as controls, as those known from Illumina or Nanopore data, are often not considered as contaminants and also not appropriately removed during analyses. Additionally, removing human host DNA may be necessary for data protection and ethical considerations to ensure that individuals cannot be identified. We developed CLEAN, a pipeline to remove unwanted sequences from both long- and short-read sequencing techniques. While focusing on Illumina and Nanopore data with their technology-specific control sequences, the pipeline can also be used for host decontamination of metagenomic reads and assemblies, or the removal of rRNA from RNA-Seq data. The results are the purified sequences and sequences identified as contaminated with statistics summarized in a report. The output can be used directly in subsequent analyses, resulting in faster computations and improved results. Although decontamination seems mundane, many contaminants are routinely overlooked, cleaned by steps that are not fully reproducible or difficult to trace. CLEAN facilitates reproducible, platform-independent data analysis in genomics and transcriptomics and is freely available at https://github.com/rki-mf1/clean under a BSD3 license.},
keywords = {assembly, metagenomics, nanopore, RNA / transcriptomics, software},
pubstate = {published},
tppubtype = {article}
}
Many biological and medical questions are answered based on the analysis of sequence data. However, we can find contamination, artificial spike-ins, and overrepresented rRNA (ribosomal RNA) sequences in various read collections and assemblies. In particular, spike-ins used as controls, as those known from Illumina or Nanopore data, are often not considered as contaminants and also not appropriately removed during analyses. Additionally, removing human host DNA may be necessary for data protection and ethical considerations to ensure that individuals cannot be identified. We developed CLEAN, a pipeline to remove unwanted sequences from both long- and short-read sequencing techniques. While focusing on Illumina and Nanopore data with their technology-specific control sequences, the pipeline can also be used for host decontamination of metagenomic reads and assemblies, or the removal of rRNA from RNA-Seq data. The results are the purified sequences and sequences identified as contaminated with statistics summarized in a report. The output can be used directly in subsequent analyses, resulting in faster computations and improved results. Although decontamination seems mundane, many contaminants are routinely overlooked, cleaned by steps that are not fully reproducible or difficult to trace. CLEAN facilitates reproducible, platform-independent data analysis in genomics and transcriptomics and is freely available at https://github.com/rki-mf1/clean under a BSD3 license.
Meyer, Daria; Barth, Emanuel; Wiehle, Laura; Marz, Manja
diffONT: predicting methylation-specific PCR biomarkers based on nanopore sequencing data for clinical application Journal Article
In: bioRxiv, 2025.
Abstract | Links | BibTeX | Tags: cancer, DNA / genomics, nanopore, nucleic acid modifications, software
@article{nokey_73,
title = {diffONT: predicting methylation-specific PCR biomarkers based on nanopore sequencing data for clinical application},
author = {Daria Meyer and Emanuel Barth and Laura Wiehle and Manja Marz},
doi = {10.1101/2025.02.17.638597},
year = {2025},
date = {2025-02-20},
urldate = {2025-02-20},
journal = {bioRxiv},
abstract = {DNA methylation is known to act as biomarker applicable for clinical diagnostics, especially in cancer detection. Methylation-specific PCR (MSP) is a widely used approach to screen patient samples fast and efficiently for differential methylation. During MSP, methylated regions are selectively amplified with specific primers. With nanopore sequencing, knowledge about DNA methylation is generated during direct DNA sequencing, without any need for pretreatment of the DNA. Multiple methods, mainly developed for whole-genome bisulfite sequencing (WGBS) data, exist to predict differentially methylated regions (DMRs) in the genome. However, the predicted DMRs are often very large, and not sufficiently discriminating to generate meaningful results in MSP creating a gap between theoretical cancer marker research and practical application, as no tool currently provides methylation difference predictions tailored for PCR-based diagnostics. Here we present diffONT, which predicts differentially methylated primer regions, directly suitable for MSP primer design and thus allowing a direct translation into practical approaches. diffONT takes into account (i) the specific length of primer and amplicon regions, (ii) the fact that one condition should be unmethylated, and (iii) a minimal required amount of differentially methylated cytosines within the primer regions. Based on two nanopore sequencing data sets we compared the results of diffONT to metilene, DSS and pycoMeth. We show that the regions predicted by diffONT are more specific towards hypermethylated regions and more usable for MSP. diffONT accelerates the design of methylation-specific diagnostic assays, bridging the gap between theoretical research and clinical application.Competing Interest Statement. The authors have declared no competing interest.},
keywords = {cancer, DNA / genomics, nanopore, nucleic acid modifications, software},
pubstate = {published},
tppubtype = {article}
}
DNA methylation is known to act as biomarker applicable for clinical diagnostics, especially in cancer detection. Methylation-specific PCR (MSP) is a widely used approach to screen patient samples fast and efficiently for differential methylation. During MSP, methylated regions are selectively amplified with specific primers. With nanopore sequencing, knowledge about DNA methylation is generated during direct DNA sequencing, without any need for pretreatment of the DNA. Multiple methods, mainly developed for whole-genome bisulfite sequencing (WGBS) data, exist to predict differentially methylated regions (DMRs) in the genome. However, the predicted DMRs are often very large, and not sufficiently discriminating to generate meaningful results in MSP creating a gap between theoretical cancer marker research and practical application, as no tool currently provides methylation difference predictions tailored for PCR-based diagnostics. Here we present diffONT, which predicts differentially methylated primer regions, directly suitable for MSP primer design and thus allowing a direct translation into practical approaches. diffONT takes into account (i) the specific length of primer and amplicon regions, (ii) the fact that one condition should be unmethylated, and (iii) a minimal required amount of differentially methylated cytosines within the primer regions. Based on two nanopore sequencing data sets we compared the results of diffONT to metilene, DSS and pycoMeth. We show that the regions predicted by diffONT are more specific towards hypermethylated regions and more usable for MSP. diffONT accelerates the design of methylation-specific diagnostic assays, bridging the gap between theoretical research and clinical application.Competing Interest Statement. The authors have declared no competing interest.
Meyer, Daria; Göttsch, Winfried; Spangenberg, Jannes; Stieber, Bettina; Krautwurst, Sebastian; Hoelzer, Martin; Brandt, Christian; Linde, Joerg; zu Siederdissen, Christian Höner; Srivastava, Akash; Zarkovic, Milena; Wollny, Damian; Marz, Manja
Unlocking the Full Potential of Nanopore Sequencing: Tips, Tricks, and Advanced Data Analysis Techniques Journal Article
In: bioRxiv, 2025.
Abstract | Links | BibTeX | Tags: differential expression analysis, DNA / genomics, nanopore, nucleic acid modifications
@article{nokey,
title = {Unlocking the Full Potential of Nanopore Sequencing: Tips, Tricks, and Advanced Data Analysis Techniques},
author = {Daria Meyer and Winfried Göttsch and Jannes Spangenberg and Bettina Stieber and Sebastian Krautwurst and Martin Hoelzer and Christian Brandt and Joerg Linde and Christian {Höner zu Siederdissen} and Akash Srivastava and Milena Zarkovic and Damian Wollny and Manja Marz},
doi = {10.1101/2023.12.06.570356},
year = {2025},
date = {2025-01-27},
urldate = {2025-01-27},
journal = {bioRxiv},
abstract = {Nucleic acid sequencing is the process of identifying the sequence of DNA or RNA, with DNA used for genomes and RNA for transcriptomes. Deciphering this information has the potential to greatly advance our understanding of genomic features and cellular functions. In comparison to other available sequencing methods, nanopore sequencing stands out due to its unique advantages of processing long nucleic acid strands in real time, within a small portable device, enabling the rapid analysis of samples in diverse settings. Evolving over the past decade, nanopore sequencing remains in a state of ongoing development and refinement, resulting in persistent challenges in protocols and technology. This article employs an interdisciplinary approach, evaluating experimental and computational methods to address critical gaps in our understanding in order to maximize the information gain from this advancing technology. Here we present both overview and analysis of all aspects of nanopore sequencing by providing statistically supported insights. Thus, we aim to provide fresh perspectives on nanopore sequencing and give comprehensive guidelines for the diverse challenges that frequently impede optimal experimental outcomes.},
keywords = {differential expression analysis, DNA / genomics, nanopore, nucleic acid modifications},
pubstate = {published},
tppubtype = {article}
}
Nucleic acid sequencing is the process of identifying the sequence of DNA or RNA, with DNA used for genomes and RNA for transcriptomes. Deciphering this information has the potential to greatly advance our understanding of genomic features and cellular functions. In comparison to other available sequencing methods, nanopore sequencing stands out due to its unique advantages of processing long nucleic acid strands in real time, within a small portable device, enabling the rapid analysis of samples in diverse settings. Evolving over the past decade, nanopore sequencing remains in a state of ongoing development and refinement, resulting in persistent challenges in protocols and technology. This article employs an interdisciplinary approach, evaluating experimental and computational methods to address critical gaps in our understanding in order to maximize the information gain from this advancing technology. Here we present both overview and analysis of all aspects of nanopore sequencing by providing statistically supported insights. Thus, we aim to provide fresh perspectives on nanopore sequencing and give comprehensive guidelines for the diverse challenges that frequently impede optimal experimental outcomes.
2024
Spangenberg, Jannes; Mündnich, Stefan; Busch, Anne; Pastore, Stefan; Wierczeiko, Anna; Goettsch, Winfried; Dietrich, Vincent; Pryszcz, Leszek P.; Cruciani, Sonia; Novoa, Eva Maria; Joshi, Kandarp; Perera, Ranjan; Giorgio, Salvatore Di; Arrubarrena, Paola; Tellioglu, Irem; Poon, Chi-Lam; Wan, Yuk Kei; Göke, Jonathan; Hildebrandt, Andreas; Dieterich, Christoph; Helm, Mark; Marz, Manja; Gerber, Susanne; Alagna, Nicolo
The RMaP challenge of predicting RNA modifications by nanopore sequencing Journal Article
In: Communications Chemistry, vol. 8, iss. 1, 2024.
Abstract | Links | BibTeX | Tags: machine learning, nanopore, nucleic acid modifications, RNA / transcriptomics
@article{nokey_79,
title = {The RMaP challenge of predicting RNA modifications by nanopore sequencing},
author = {Jannes Spangenberg and Stefan Mündnich and Anne Busch and Stefan Pastore and Anna Wierczeiko and Winfried Goettsch and Vincent Dietrich and Leszek P. Pryszcz and Sonia Cruciani and Eva Maria Novoa and Kandarp Joshi and Ranjan Perera and Salvatore Di Giorgio and Paola Arrubarrena and Irem Tellioglu and Chi-Lam Poon and Yuk Kei Wan and Jonathan Göke and Andreas Hildebrandt and Christoph Dieterich and Mark Helm and Manja Marz and Susanne Gerber and Nicolo Alagna},
doi = {10.1038/s42004-025-01507-0},
year = {2024},
date = {2024-12-04},
urldate = {2024-12-04},
journal = {Communications Chemistry},
volume = {8},
issue = {1},
abstract = {The field of epitranscriptomics is undergoing a technology-driven revolution. During past decades, RNA modifications like N6-methyladenosine (m6A), pseudouridine (ψ), and 5-methylcytosine (m5C) became acknowledged for playing critical roles in cellular processes. Direct RNA sequencing by Oxford Nanopore Technologies (ONT) enabled the detection of modifications in native RNA, by detecting noncanonical RNA nucleosides properties in raw data. Consequently, the field’s cutting edge has a heavy component in computer science, opening new avenues of cooperation across the community, as exchanging data is as impactful as exchanging samples. Therefore, we seize the occasion to bring scientists together within the RNA Modification and Processing (RMaP) challenge to advance solutions for RNA modification detection and discuss ideas, problems and approaches. We show several computational methods to detect the most researched mRNA modifications (m6A, ψ, and m5C). Results demonstrate that a low prediction error and a high prediction accuracy can be achieved on these modifications across different approaches and algorithms. The RMaP challenge marks a substantial step towards improving algorithms’ comparability, reliability, and consistency in RNA modification prediction. It points out the deficits in this young field that need to be addressed in further challenges.},
keywords = {machine learning, nanopore, nucleic acid modifications, RNA / transcriptomics},
pubstate = {published},
tppubtype = {article}
}
The field of epitranscriptomics is undergoing a technology-driven revolution. During past decades, RNA modifications like N6-methyladenosine (m6A), pseudouridine (ψ), and 5-methylcytosine (m5C) became acknowledged for playing critical roles in cellular processes. Direct RNA sequencing by Oxford Nanopore Technologies (ONT) enabled the detection of modifications in native RNA, by detecting noncanonical RNA nucleosides properties in raw data. Consequently, the field’s cutting edge has a heavy component in computer science, opening new avenues of cooperation across the community, as exchanging data is as impactful as exchanging samples. Therefore, we seize the occasion to bring scientists together within the RNA Modification and Processing (RMaP) challenge to advance solutions for RNA modification detection and discuss ideas, problems and approaches. We show several computational methods to detect the most researched mRNA modifications (m6A, ψ, and m5C). Results demonstrate that a low prediction error and a high prediction accuracy can be achieved on these modifications across different approaches and algorithms. The RMaP challenge marks a substantial step towards improving algorithms’ comparability, reliability, and consistency in RNA modification prediction. It points out the deficits in this young field that need to be addressed in further challenges.
zu Siederdissen, Christian Höner; Spangenberg, Jannes; Bisdorf, Kevin; Krautwurst, Sebastian; Srivastava, Akash; Marz, Manja; Taubert, Martin
Nanopore sequencing enables novel detection of deuterium incorporation in DNA Journal Article
In: Computational and Structural Biotechnology Journal, vol. 23, 2024.
Abstract | Links | BibTeX | Tags: bacteria, DNA / genomics, machine learning, metagenomics, nanopore, nucleic acid modifications
@article{nokey_74,
title = {Nanopore sequencing enables novel detection of deuterium incorporation in DNA},
author = {Christian {Höner zu Siederdissen} and Jannes Spangenberg and Kevin Bisdorf and Sebastian Krautwurst and Akash Srivastava and Manja Marz and Martin Taubert},
doi = {10.1016/j.csbj.2024.09.027},
year = {2024},
date = {2024-10-03},
urldate = {2024-10-03},
journal = {Computational and Structural Biotechnology Journal},
volume = {23},
abstract = {Identifying active microbes is crucial to understand their role in ecosystem functions. Metabolic labeling with heavy, non-radioactive isotopes, i.e., stable isotope probing (SIP), can track active microbes by detecting heavy isotope incorporation in biomolecules such as DNA. However, the detection of heavy isotope-labeled nucleotides directly during sequencing has, to date, not been achieved. In this study, Oxford nanopore sequencing was utilized to detect heavy isotopes incorporation in DNA molecules. Two isotopes widely used in SIP experiments were employed to label a bacterial isolate: deuterium (D, as D2O) and carbon-13 (13C, as glucose). We hypothesize that labeled DNA is distinguishable from unlabeled DNA by changes in the nanopore signal. To verify this distinction, we employed a Bayesian classifier trained on signal distributions of short oligonucleotides (k-mers) from labeled and unlabeled sequencing reads. Our results show a clear distinction between D-labeled and unlabeled reads, based on changes in median and median absolute deviation (MAD) of the nanopore signals for different k-mers. In contrast, 13C-labeled DNA cannot be distinguished from unlabeled DNA. For D, the model employed correctly predicted more than 85% of the reads. Even when metabolic labeling was conducted with only 30% D2O, 80% of the obtained reads were correctly classified with a 5% false discovery rate. Our work demonstrates the feasibility of direct detection of deuterium incorporation in DNA molecules during Oxford nanopore sequencing. This finding represents a first step in establishing the combined use of nanopore sequencing and SIP for tracking active organisms in microbial ecology.},
keywords = {bacteria, DNA / genomics, machine learning, metagenomics, nanopore, nucleic acid modifications},
pubstate = {published},
tppubtype = {article}
}
Identifying active microbes is crucial to understand their role in ecosystem functions. Metabolic labeling with heavy, non-radioactive isotopes, i.e., stable isotope probing (SIP), can track active microbes by detecting heavy isotope incorporation in biomolecules such as DNA. However, the detection of heavy isotope-labeled nucleotides directly during sequencing has, to date, not been achieved. In this study, Oxford nanopore sequencing was utilized to detect heavy isotopes incorporation in DNA molecules. Two isotopes widely used in SIP experiments were employed to label a bacterial isolate: deuterium (D, as D2O) and carbon-13 (13C, as glucose). We hypothesize that labeled DNA is distinguishable from unlabeled DNA by changes in the nanopore signal. To verify this distinction, we employed a Bayesian classifier trained on signal distributions of short oligonucleotides (k-mers) from labeled and unlabeled sequencing reads. Our results show a clear distinction between D-labeled and unlabeled reads, based on changes in median and median absolute deviation (MAD) of the nanopore signals for different k-mers. In contrast, 13C-labeled DNA cannot be distinguished from unlabeled DNA. For D, the model employed correctly predicted more than 85% of the reads. Even when metabolic labeling was conducted with only 30% D2O, 80% of the obtained reads were correctly classified with a 5% false discovery rate. Our work demonstrates the feasibility of direct detection of deuterium incorporation in DNA molecules during Oxford nanopore sequencing. This finding represents a first step in establishing the combined use of nanopore sequencing and SIP for tracking active organisms in microbial ecology.
2023
Triebel, Sandra; Sachse, Konrad; Weber, Michael; Heller, Martin; Diezel, Celia; Hölzer, Martin; Schnee, Christiane; Marz, Manja
De novo genome assembly resolving repetitive structures enables genomic analysis of 35 European Mycoplasmopsis bovis strains Journal Article
In: BMC Genomics, vol. 24, iss. 1, no. 548, 2023, ISBN: 1471-2164.
Abstract | Links | BibTeX | Tags: assembly, bacteria, DNA / genomics, nanopore, phylogenetics
@article{nokey_44,
title = {\textit{De novo} genome assembly resolving repetitive structures enables genomic analysis of 35 European \textit{Mycoplasmopsis bovis} strains},
author = {Sandra Triebel and Konrad Sachse and Michael Weber and Martin Heller and Celia Diezel and Martin Hölzer and Christiane Schnee and Manja Marz },
doi = {10.1186/s12864-023-09618-5},
isbn = {1471-2164},
year = {2023},
date = {2023-09-16},
urldate = {2023-09-16},
journal = {BMC Genomics},
volume = {24},
number = {548},
issue = {1},
abstract = {Mycoplasmopsis (M.) bovis, the agent of mastitis, pneumonia, and arthritis in cattle, harbors a small genome of approximately 1 Mbp. Combining data from Illumina and Nanopore technologies, we sequenced and assembled the genomes of 35 European strains and isolate DL422_88 from Cuba. While the high proportion of repetitive structures in M. bovis genomes represent a particular challenge, implementation of our own pipeline Mycovista (available on GitHub www.github.com/sandraTriebel/mycovista ) in a hybrid approach enabled contiguous assembly of the genomes and, consequently, improved annotation rates considerably. To put our European strain panel in a global context, we analyzed the new genome sequences together with 175 genome assemblies from public databases. Construction of a phylogenetic tree based on core genes of these 219 strains revealed a clustering pattern according to geographical origin, with European isolates positioned on clades 4 and 5. Genomic data allowing assignment of strains to tissue specificity or certain disease manifestations could not be identified. Seven strains isolated from cattle with systemic circular condition (SCC), still a largely unknown manifestation of M. bovis disease, were located on both clades 4 and 5. Pairwise association analysis revealed 108 genomic elements associated with a particular clade of the phylogenetic tree. Further analyzing these hits, 25 genes are functionally annotated and could be linked to a M. bovis protein, e.g. various proteases and nucleases, as well as ten variable surface lipoproteins (Vsps) and other surface proteins. These clade-specific genes could serve as useful markers in epidemiological and clinical surveys.},
keywords = {assembly, bacteria, DNA / genomics, nanopore, phylogenetics},
pubstate = {published},
tppubtype = {article}
}
Mycoplasmopsis (M.) bovis, the agent of mastitis, pneumonia, and arthritis in cattle, harbors a small genome of approximately 1 Mbp. Combining data from Illumina and Nanopore technologies, we sequenced and assembled the genomes of 35 European strains and isolate DL422_88 from Cuba. While the high proportion of repetitive structures in M. bovis genomes represent a particular challenge, implementation of our own pipeline Mycovista (available on GitHub www.github.com/sandraTriebel/mycovista ) in a hybrid approach enabled contiguous assembly of the genomes and, consequently, improved annotation rates considerably. To put our European strain panel in a global context, we analyzed the new genome sequences together with 175 genome assemblies from public databases. Construction of a phylogenetic tree based on core genes of these 219 strains revealed a clustering pattern according to geographical origin, with European isolates positioned on clades 4 and 5. Genomic data allowing assignment of strains to tissue specificity or certain disease manifestations could not be identified. Seven strains isolated from cattle with systemic circular condition (SCC), still a largely unknown manifestation of M. bovis disease, were located on both clades 4 and 5. Pairwise association analysis revealed 108 genomic elements associated with a particular clade of the phylogenetic tree. Further analyzing these hits, 25 genes are functionally annotated and could be linked to a M. bovis protein, e.g. various proteases and nucleases, as well as ten variable surface lipoproteins (Vsps) and other surface proteins. These clade-specific genes could serve as useful markers in epidemiological and clinical surveys.
Thomas, Christine; Methner, Ulrich; Marz, Manja; Linde, Jörg
In: Frontiers in Veterinary Science, vol. 10, 2023, ISBN: 2297-1769.
Abstract | Links | BibTeX | Tags: bacteria, nanopore
@article{nokey_40,
title = {Oxford nanopore technologies-a valuable tool to generate whole-genome sequencing data for \textit{in silico} serotyping and the detection of genetic markers in \textit{Salmonella}},
author = {Christine Thomas and Ulrich Methner and Manja Marz and Jörg Linde},
doi = {10.3389/fvets.2023.1178922},
isbn = {2297-1769},
year = {2023},
date = {2023-06-01},
urldate = {2023-06-01},
journal = {Frontiers in Veterinary Science},
volume = {10},
abstract = {Bacteria of the genus Salmonella pose a major risk to livestock, the food economy, and public health. Salmonella infections are one of the leading causes of food poisoning. The identification of serovars of Salmonella achieved by their diverse surface antigens is essential to gain information on their epidemiological context. Traditionally, slide agglutination has been used for serotyping. In recent years, whole-genome sequencing (WGS) followed by in silico serotyping has been established as an alternative method for serotyping and the detection of genetic markers for Salmonella. Until now, WGS data generated with Illumina sequencing are used to validate in silico serotyping methods. Oxford Nanopore Technologies (ONT) opens the possibility to sequence ultra-long reads and has frequently been used for bacterial sequencing. In this study, ONT sequencing data of 28 Salmonella strains of different serovars with epidemiological relevance in humans, food, and animals were taken to investigate the performance of the in silico serotyping tools SISTR and SeqSero2 compared to traditional slide agglutination tests. Moreover, the detection of genetic markers for resistance against antimicrobial agents, virulence, and plasmids was studied by comparing WGS data based on ONT with WGS data based on Illumina. Based on the ONT data from flow cell version R9.4.1, in silico serotyping achieved an accuracy of 96.4 and 92% for the tools SISTR and SeqSero2, respectively. Highly similar sets of genetic markers comparing both sequencing technologies were identified. Taking the ongoing improvement of basecalling and flow cells into account, ONT data can be used for Salmonella in silico serotyping and genetic marker detection.},
keywords = {bacteria, nanopore},
pubstate = {published},
tppubtype = {article}
}
Bacteria of the genus Salmonella pose a major risk to livestock, the food economy, and public health. Salmonella infections are one of the leading causes of food poisoning. The identification of serovars of Salmonella achieved by their diverse surface antigens is essential to gain information on their epidemiological context. Traditionally, slide agglutination has been used for serotyping. In recent years, whole-genome sequencing (WGS) followed by in silico serotyping has been established as an alternative method for serotyping and the detection of genetic markers for Salmonella. Until now, WGS data generated with Illumina sequencing are used to validate in silico serotyping methods. Oxford Nanopore Technologies (ONT) opens the possibility to sequence ultra-long reads and has frequently been used for bacterial sequencing. In this study, ONT sequencing data of 28 Salmonella strains of different serovars with epidemiological relevance in humans, food, and animals were taken to investigate the performance of the in silico serotyping tools SISTR and SeqSero2 compared to traditional slide agglutination tests. Moreover, the detection of genetic markers for resistance against antimicrobial agents, virulence, and plasmids was studied by comparing WGS data based on ONT with WGS data based on Illumina. Based on the ONT data from flow cell version R9.4.1, in silico serotyping achieved an accuracy of 96.4 and 92% for the tools SISTR and SeqSero2, respectively. Highly similar sets of genetic markers comparing both sequencing technologies were identified. Taking the ongoing improvement of basecalling and flow cells into account, ONT data can be used for Salmonella in silico serotyping and genetic marker detection.
Erkes, Annett; Grove, René P; Žarković, Milena; Krautwurst, Sebastian; Koebnik, Ralf; Morgan, Richard D; Wilson, Geoffrey G; Hölzer, Martin; Marz, Manja; Boch, Jens; Grau, Jan
Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing Journal Article
In: BMC Genomics, vol. 24, iss. 1, pp. 151, 2023.
Abstract | Links | BibTeX | Tags: assembly, DNA / genomics, nanopore
@article{nokey,
title = {Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing},
author = {Annett Erkes and René P Grove and Milena Žarković and Sebastian Krautwurst and Ralf Koebnik and Richard D Morgan and Geoffrey G Wilson and Martin Hölzer and Manja Marz and Jens Boch and Jan Grau
},
doi = {10.1186/s12864-023-09228-1},
year = {2023},
date = {2023-03-27},
journal = {BMC Genomics},
volume = {24},
issue = {1},
pages = {151},
abstract = {Background: Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches.
Results: Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads.
Conclusions: Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.},
keywords = {assembly, DNA / genomics, nanopore},
pubstate = {published},
tppubtype = {article}
}
Background: Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches.
Results: Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads.
Conclusions: Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.
Results: Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads.
Conclusions: Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.
Spangenberg, Jannes; zu Siederdissen, Christian Höner; Žarković, Milena; Triebel, Sandra; Rose, Ruben; Christophersen, Christina Martínez; Paltzow, Lea; Hegab, Mohsen M.; Wansorra, Anna; Srivastava, Akash; Krumbholz, Andi; Marz, Manja
Magnipore: Prediction of differential single nucleotide changes in the Oxford Nanopore Technologies sequencing signal of SARS-CoV-2 samples Journal Article
In: bioRxiv, 2023.
Abstract | Links | BibTeX | Tags: coronavirus, nanopore, nucleic acid modifications, RNA / transcriptomics, software, viruses
@article{nokey,
title = {Magnipore: Prediction of differential single nucleotide changes in the Oxford Nanopore Technologies sequencing signal of SARS-CoV-2 samples},
author = {Jannes Spangenberg and Christian {Höner zu Siederdissen} and Milena Žarković and Sandra Triebel and Ruben Rose and Christina Martínez Christophersen and Lea Paltzow and Mohsen M. Hegab and Anna Wansorra and Akash Srivastava and Andi Krumbholz and Manja Marz},
doi = {10.1101/2023.03.17.533105},
year = {2023},
date = {2023-03-17},
urldate = {2023-03-17},
journal = {bioRxiv},
abstract = {Oxford Nanopore Technologies (ONT) allows direct sequencing of ribonucleic acids (RNA) and, in addition, detection of possible RNA modifications due to deviations from the expected ONT signal. The software available so far for this purpose can only detect a small number of modifications. Alternatively, two samples can be compared for different RNA modifications. We present Magnipore, a novel tool to search for significant signal shifts between samples of Oxford Nanopore data from similar or related species. Magnipore classifies them into mutations and potential modifications. We use Magnipore to compare SARS-CoV-2 samples. Included were representatives of the early 2020s Pango lineages (n=6), samples from Pango lineages B.1.1.7 (n=2, Alpha), B.1.617.2 (n=1, Delta), and B.1.529 (n=7, Omicron). Magnipore utilizes position-wise Gaussian distribution models and a comprehensible significance threshold to find differential signals. In the case of Alpha and Delta, Magnipore identifies 55 detected mutations and 15 sites that hint at differential modifications. We predicted potential virus-variant and variant-group-specific differential modifications. Magnipore contributes to advancing RNA modification analysis in the context of viruses and virus variants.},
keywords = {coronavirus, nanopore, nucleic acid modifications, RNA / transcriptomics, software, viruses},
pubstate = {published},
tppubtype = {article}
}
Oxford Nanopore Technologies (ONT) allows direct sequencing of ribonucleic acids (RNA) and, in addition, detection of possible RNA modifications due to deviations from the expected ONT signal. The software available so far for this purpose can only detect a small number of modifications. Alternatively, two samples can be compared for different RNA modifications. We present Magnipore, a novel tool to search for significant signal shifts between samples of Oxford Nanopore data from similar or related species. Magnipore classifies them into mutations and potential modifications. We use Magnipore to compare SARS-CoV-2 samples. Included were representatives of the early 2020s Pango lineages (n=6), samples from Pango lineages B.1.1.7 (n=2, Alpha), B.1.617.2 (n=1, Delta), and B.1.529 (n=7, Omicron). Magnipore utilizes position-wise Gaussian distribution models and a comprehensible significance threshold to find differential signals. In the case of Alpha and Delta, Magnipore identifies 55 detected mutations and 15 sites that hint at differential modifications. We predicted potential virus-variant and variant-group-specific differential modifications. Magnipore contributes to advancing RNA modification analysis in the context of viruses and virus variants.
2022
Houwaart, Torsten; Belhaj, Samir; Tawalbeh, Emran; Nagels, Dirk; Fröhlich, Yara; Finzer, Patrick; Ciruela, Pilar; Sabrià, Aurora; Herrero, Mercè; Andrés, Cristina; Antón, Andrés; Benmoumene, Assia; Asskali, Dounia; Haidar, Hussein; von Dahlen, Janina; Nicolai, Jessica; Stiller, Mygg; Blum, Jacqueline; Lange, Christian; Adelmann, Carla; Schroer, Britta; Osmers, Ute; Grice, Christiane; Kirfel, Phillipp P; Jomaa, Hassan; Strelow, Daniel; Hülse, Lisanna; Pigulla, Moritz; Kreuzer, Pascal; Tyshaieva, Alona; Weber, Jonas; Wienemann, Tobias; Vasconcelos, Malte Kohns; Hoffmann, Katrin; Lübke, Nadine; Hauka, Sandra; Andree, Marcel; Scholz, Claus Jürgen; Jazmati, Nathalie; Göbels, Klaus; Zotz, Rainer; Pfeffer, Klaus; Timm, Jörg; Ehlkes, Lutz; Walker, Andreas; Dilthey, Alexander T; (DeCOI), Deutsche COVID-19 OMICS Initiative
In: Euro Surveill, vol. 27, iss. 43, pp. 2101089, 2022.
Abstract | Links | BibTeX | Tags: coronavirus, nanopore, RNA / transcriptomics, viruses
@article{nokey,
title = {Integrated genomic surveillance enables tracing of person-to-person SARS-CoV-2 transmission chains during community transmission and reveals extensive onward transmission of travel-imported infections, Germany, June to July 2021},
author = {Torsten Houwaart and Samir Belhaj and Emran Tawalbeh and Dirk Nagels and Yara Fröhlich and Patrick Finzer and Pilar Ciruela and Aurora Sabrià and Mercè Herrero and Cristina Andrés and Andrés Antón and Assia Benmoumene and Dounia Asskali and Hussein Haidar and Janina von Dahlen and Jessica Nicolai and Mygg Stiller and Jacqueline Blum and Christian Lange and Carla Adelmann and Britta Schroer and Ute Osmers and Christiane Grice and Phillipp P Kirfel and Hassan Jomaa and Daniel Strelow and Lisanna Hülse and Moritz Pigulla and Pascal Kreuzer and Alona Tyshaieva and Jonas Weber and Tobias Wienemann and Malte Kohns Vasconcelos and Katrin Hoffmann and Nadine Lübke and Sandra Hauka and Marcel Andree and Claus Jürgen Scholz and Nathalie Jazmati and Klaus Göbels and Rainer Zotz and Klaus Pfeffer and Jörg Timm and Lutz Ehlkes and Andreas Walker and Alexander T Dilthey and Deutsche COVID-19 OMICS Initiative (DeCOI)},
doi = {10.2807/1560-7917.ES.2022.27.43.2101089},
year = {2022},
date = {2022-10-27},
urldate = {2022-10-27},
journal = {Euro Surveill},
volume = {27},
issue = {43},
pages = {2101089},
abstract = {BackgroundTracking person-to-person SARS-CoV-2 transmission in the population is important to understand the epidemiology of community transmission and may contribute to the containment of SARS-CoV-2. Neither contact tracing nor genomic surveillance alone, however, are typically sufficient to achieve this objective.AimWe demonstrate the successful application of the integrated genomic surveillance (IGS) system of the German city of Düsseldorf for tracing SARS-CoV-2 transmission chains in the population as well as detecting and investigating travel-associated SARS-CoV-2 infection clusters.MethodsGenomic surveillance, phylogenetic analysis, and structured case interviews were integrated to elucidate two genetically defined clusters of SARS-CoV-2 isolates detected by IGS in Düsseldorf in July 2021.ResultsCluster 1 (n = 67 Düsseldorf cases) and Cluster 2 (n = 36) were detected in a surveillance dataset of 518 high-quality SARS-CoV-2 genomes from Düsseldorf (53% of total cases, sampled mid-June to July 2021). Cluster 1 could be traced back to a complex pattern of transmission in nightlife venues following a putative importation by a SARS-CoV-2-infected return traveller (IP) in late June; 28 SARS-CoV-2 cases could be epidemiologically directly linked to IP. Supported by viral genome data from Spain, Cluster 2 was shown to represent multiple independent introduction events of a viral strain circulating in Catalonia and other European countries, followed by diffuse community transmission in Düsseldorf.ConclusionIGS enabled high-resolution tracing of SARS-CoV-2 transmission in an internationally connected city during community transmission and provided infection chain-level evidence of the downstream propagation of travel-imported SARS-CoV-2 cases.},
keywords = {coronavirus, nanopore, RNA / transcriptomics, viruses},
pubstate = {published},
tppubtype = {article}
}
BackgroundTracking person-to-person SARS-CoV-2 transmission in the population is important to understand the epidemiology of community transmission and may contribute to the containment of SARS-CoV-2. Neither contact tracing nor genomic surveillance alone, however, are typically sufficient to achieve this objective.AimWe demonstrate the successful application of the integrated genomic surveillance (IGS) system of the German city of Düsseldorf for tracing SARS-CoV-2 transmission chains in the population as well as detecting and investigating travel-associated SARS-CoV-2 infection clusters.MethodsGenomic surveillance, phylogenetic analysis, and structured case interviews were integrated to elucidate two genetically defined clusters of SARS-CoV-2 isolates detected by IGS in Düsseldorf in July 2021.ResultsCluster 1 (n = 67 Düsseldorf cases) and Cluster 2 (n = 36) were detected in a surveillance dataset of 518 high-quality SARS-CoV-2 genomes from Düsseldorf (53% of total cases, sampled mid-June to July 2021). Cluster 1 could be traced back to a complex pattern of transmission in nightlife venues following a putative importation by a SARS-CoV-2-infected return traveller (IP) in late June; 28 SARS-CoV-2 cases could be epidemiologically directly linked to IP. Supported by viral genome data from Spain, Cluster 2 was shown to represent multiple independent introduction events of a viral strain circulating in Catalonia and other European countries, followed by diffuse community transmission in Düsseldorf.ConclusionIGS enabled high-resolution tracing of SARS-CoV-2 transmission in an internationally connected city during community transmission and provided infection chain-level evidence of the downstream propagation of travel-imported SARS-CoV-2 cases.
Fuesslin, Valeria; Krautwurst, Sebastian; Srivastava, Akash; Winter, Doris; Liedigk, Britta; Thye, Thorsten; Herrera-León, Silvia; Wohl, Shirlee; May, Jürgen; Fobil, Julius N.; Eibach, Daniel; Marz, Manja; Schuldt, Kathrin
In: Front Microbiol, vol. 13, pp. 909692, 2022.
Abstract | Links | BibTeX | Tags: bacteria, DNA / genomics, nanopore
@article{Fuesslin2022,
title = {Prediction of Antibiotic Susceptibility Profiles of \textit{Vibrio cholerae} Isolates From Whole Genome Illumina and Nanopore Sequencing Data: CholerAegon},
author = {Valeria Fuesslin and Sebastian Krautwurst and Akash Srivastava and Doris Winter and Britta Liedigk and Thorsten Thye and Silvia Herrera-León and Shirlee Wohl and Jürgen May and Julius N. Fobil and Daniel Eibach and Manja Marz and Kathrin Schuldt},
url = {https://github.com/RaverJay/CholerAegon },
doi = {10.3389/fmicb.2022.909692},
year = {2022},
date = {2022-06-22},
journal = {Front Microbiol},
volume = {13},
pages = {909692},
abstract = {During the last decades, antimicrobial resistance (AMR) has become a global public health concern. Nowadays multi-drug resistance is commonly observed in strains of Vibrio cholerae, the etiological agent of cholera. In order to limit the spread of pathogenic drug-resistant bacteria and to maintain treatment options the analysis of clinical samples and their AMR profiles are essential. Particularly, in low-resource settings a timely analysis of AMR profiles is often impaired due to lengthy culturing procedures for antibiotic susceptibility testing or lack of laboratory capacity. In this study, we explore the applicability of whole genome sequencing for the prediction of AMR profiles of V. cholerae. We developed the pipeline CholerAegon for the in silico prediction of AMR profiles of 82 V. cholerae genomes assembled from long and short sequencing reads. By correlating the predicted profiles with results from phenotypic antibiotic susceptibility testing we show that the prediction can replace in vitro susceptibility testing for five of seven antibiotics. Because of the relatively low costs, possibility for real-time data analyses, and portability, the Oxford Nanopore Technologies MinION sequencing platform—especially in light of an upcoming less error-prone technology for the platform—appears to be well suited for pathogen genomic analyses such as the one described here. Together with CholerAegon, it can leverage pathogen genomics to improve disease surveillance and to control further spread of antimicrobial resistance.},
keywords = {bacteria, DNA / genomics, nanopore},
pubstate = {published},
tppubtype = {article}
}
During the last decades, antimicrobial resistance (AMR) has become a global public health concern. Nowadays multi-drug resistance is commonly observed in strains of Vibrio cholerae, the etiological agent of cholera. In order to limit the spread of pathogenic drug-resistant bacteria and to maintain treatment options the analysis of clinical samples and their AMR profiles are essential. Particularly, in low-resource settings a timely analysis of AMR profiles is often impaired due to lengthy culturing procedures for antibiotic susceptibility testing or lack of laboratory capacity. In this study, we explore the applicability of whole genome sequencing for the prediction of AMR profiles of V. cholerae. We developed the pipeline CholerAegon for the in silico prediction of AMR profiles of 82 V. cholerae genomes assembled from long and short sequencing reads. By correlating the predicted profiles with results from phenotypic antibiotic susceptibility testing we show that the prediction can replace in vitro susceptibility testing for five of seven antibiotics. Because of the relatively low costs, possibility for real-time data analyses, and portability, the Oxford Nanopore Technologies MinION sequencing platform—especially in light of an upcoming less error-prone technology for the platform—appears to be well suited for pathogen genomic analyses such as the one described here. Together with CholerAegon, it can leverage pathogen genomics to improve disease surveillance and to control further spread of antimicrobial resistance.
Barth, Emanuel; Burggraaff, Johannes; Srivastava, Akash; Winckler, Thomas
Nanopore sequencing for mapping of retrotransposon integration sites in the Dictyostelium discoideum genome Journal Article
In: MicroPubl Biol, 2022.
Abstract | Links | BibTeX | Tags: DNA / genomics, nanopore
@article{nokey,
title = {Nanopore sequencing for mapping of retrotransposon integration sites in the \textit{Dictyostelium discoideum} genome},
author = {Emanuel Barth and Johannes Burggraaff and Akash Srivastava and Thomas Winckler
},
doi = {10.17912/micropub.biology.000543},
year = {2022},
date = {2022-03-18},
journal = {MicroPubl Biol},
abstract = {The unicellular eukaryote \textit{Dictyostelium discoideum} has a gene-dense haploid genome. This configuration presents mobile elements with the particular challenge of replicating without causing excessive damage to the host through insertional mutagenesis or recombination between repetitive sequences. \textit{D. discoideum} harbors an active population of the retrotransposon TRE5-A that integrates in a narrow window of ~50 bp upstream of tRNA genes. We assume that this integration preference was developed to avoid the disruption of protein-coding genes. Therefore, we recently mapped new integrations of a genetically tagged TRE5-A element at tRNA genes using PCR-based enrichment of integration junctions. However, the PCR-based enrichment produced several artificial DNA fusions that prevented the mapping of integration sites in unknown places of the genome. Here, we reanalyzed the previous experiment using nanopore sequencing. We summarize the advantages and limitations of direct genome resequencing for the mapping of mobile element integrations.},
keywords = {DNA / genomics, nanopore},
pubstate = {published},
tppubtype = {article}
}
The unicellular eukaryote Dictyostelium discoideum has a gene-dense haploid genome. This configuration presents mobile elements with the particular challenge of replicating without causing excessive damage to the host through insertional mutagenesis or recombination between repetitive sequences. D. discoideum harbors an active population of the retrotransposon TRE5-A that integrates in a narrow window of ~50 bp upstream of tRNA genes. We assume that this integration preference was developed to avoid the disruption of protein-coding genes. Therefore, we recently mapped new integrations of a genetically tagged TRE5-A element at tRNA genes using PCR-based enrichment of integration junctions. However, the PCR-based enrichment produced several artificial DNA fusions that prevented the mapping of integration sites in unknown places of the genome. Here, we reanalyzed the previous experiment using nanopore sequencing. We summarize the advantages and limitations of direct genome resequencing for the mapping of mobile element integrations.
2021
Martín-Hernández, Giselle C; Müller, Bettina; Chmielarz, Mikołaj; Brandt, Christian; Hölzer, Martin; Viehweger, Adrian; Passoth, Volkmar
Chromosome-level genome assembly and transcriptome-based annotation of the oleaginous yeast Rhodotorula toruloides CBS 14 Journal Article
In: Genomics, vol. 113, no. 6, pp. 4022-4027, 2021.
Abstract | Links | BibTeX | Tags: annotation, assembly, DNA / genomics, fungi, nanopore
@article{Martín-Hernández2021,
title = {Chromosome-level genome assembly and transcriptome-based annotation of the oleaginous yeast Rhodotorula toruloides CBS 14},
author = {Giselle C Martín-Hernández and Bettina Müller and Mikołaj Chmielarz and Christian Brandt and Martin Hölzer and Adrian Viehweger and Volkmar Passoth
},
doi = {10.1016/j.ygeno.2021.10.006},
year = {2021},
date = {2021-10-11},
urldate = {2021-10-11},
journal = {Genomics},
volume = {113},
number = {6},
pages = {4022-4027},
abstract = {Rhodotorula toruloides is an oleaginous yeast with high biotechnological potential. In order to understand the molecular physiology of lipid synthesis in R. toruloides and to advance metabolic engineering, a high-resolution genome is required. We constructed a genome draft of R. toruloides CBS 14, using a hybrid assembly approach, consisting of short and long reads generated by Illumina and Nanopore sequencing, respectively. The genome draft consists of 23 contigs and 3 scaffolds, with a N50 length of 1,529,952 bp, thus largely representing chromosomal organization. The total size of the genome is 20,534,857 bp and the overall GC content is 61.83%. Transcriptomic data from different growth conditions was used to aid species-specific gene annotation. We annotated 9464 genes and identified 11,691 transcripts. Furthermore, we demonstrated the presence of a potential plasmid, an extrachromosomal circular structure of about 11 kb with a copy number about three times as high as the other chromosomes.},
keywords = {annotation, assembly, DNA / genomics, fungi, nanopore},
pubstate = {published},
tppubtype = {article}
}
Rhodotorula toruloides is an oleaginous yeast with high biotechnological potential. In order to understand the molecular physiology of lipid synthesis in R. toruloides and to advance metabolic engineering, a high-resolution genome is required. We constructed a genome draft of R. toruloides CBS 14, using a hybrid assembly approach, consisting of short and long reads generated by Illumina and Nanopore sequencing, respectively. The genome draft consists of 23 contigs and 3 scaffolds, with a N50 length of 1,529,952 bp, thus largely representing chromosomal organization. The total size of the genome is 20,534,857 bp and the overall GC content is 61.83%. Transcriptomic data from different growth conditions was used to aid species-specific gene annotation. We annotated 9464 genes and identified 11,691 transcripts. Furthermore, we demonstrated the presence of a potential plasmid, an extrachromosomal circular structure of about 11 kb with a copy number about three times as high as the other chromosomes.
Brandt, Christian; Krautwurst, Sebastian; Spott, Riccardo; Lohde, Mara; Jundzill, Mateusz; Marquet, Mike; Hölzer, Martin
poreCov - An Easy to Use, Fast, and Robust Workflow for SARS-CoV-2 Genome Reconstruction via Nanopore Sequencing Journal Article
In: Front Genet, vol. 12, pp. 711437, 2021.
Abstract | Links | BibTeX | Tags: coronavirus, nanopore, RNA / transcriptomics, software, viruses
@article{Brandt2021,
title = {poreCov - An Easy to Use, Fast, and Robust Workflow for SARS-CoV-2 Genome Reconstruction via Nanopore Sequencing},
author = {Christian Brandt and Sebastian Krautwurst and Riccardo Spott and Mara Lohde and Mateusz Jundzill and Mike Marquet and Martin Hölzer},
url = {https://github.com/replikation/poreCov},
doi = {10.3389/fgene.2021.711437},
year = {2021},
date = {2021-07-28},
urldate = {2021-07-28},
journal = {Front Genet},
volume = {12},
pages = {711437},
abstract = {In response to the SARS-CoV-2 pandemic, a highly increased sequencing effort has been established worldwide to track and trace ongoing viral evolution. Technologies, such as nanopore sequencing via the ARTIC protocol are used to reliably generate genomes from raw sequencing data as a crucial base for molecular surveillance. However, for many labs that perform SARS-CoV-2 sequencing, bioinformatics is still a major bottleneck, especially if hundreds of samples need to be processed in a recurring fashion. Pipelines developed for short-read data cannot be applied to nanopore data. Therefore, specific long-read tools and parameter settings need to be orchestrated to enable accurate genotyping and robust reference-based genome reconstruction of SARS-CoV-2 genomes from nanopore data. Here we present poreCov, a highly parallel workflow written in Nextflow, using containers to wrap all the tools necessary for a routine SARS-CoV-2 sequencing lab into one program. The ease of installation, combined with concise summary reports that clearly highlight all relevant information, enables rapid and reliable analysis of hundreds of SARS-CoV-2 raw sequence data sets or genomes. poreCov is freely available on GitHub under the GNUv3 license: github.com/replikation/poreCov.},
keywords = {coronavirus, nanopore, RNA / transcriptomics, software, viruses},
pubstate = {published},
tppubtype = {article}
}
In response to the SARS-CoV-2 pandemic, a highly increased sequencing effort has been established worldwide to track and trace ongoing viral evolution. Technologies, such as nanopore sequencing via the ARTIC protocol are used to reliably generate genomes from raw sequencing data as a crucial base for molecular surveillance. However, for many labs that perform SARS-CoV-2 sequencing, bioinformatics is still a major bottleneck, especially if hundreds of samples need to be processed in a recurring fashion. Pipelines developed for short-read data cannot be applied to nanopore data. Therefore, specific long-read tools and parameter settings need to be orchestrated to enable accurate genotyping and robust reference-based genome reconstruction of SARS-CoV-2 genomes from nanopore data. Here we present poreCov, a highly parallel workflow written in Nextflow, using containers to wrap all the tools necessary for a routine SARS-CoV-2 sequencing lab into one program. The ease of installation, combined with concise summary reports that clearly highlight all relevant information, enables rapid and reliable analysis of hundreds of SARS-CoV-2 raw sequence data sets or genomes. poreCov is freely available on GitHub under the GNUv3 license: github.com/replikation/poreCov.
Walker, Andreas; Houwaart, Torsten; Finzer, Patrick; Ehlkes, Lutz; Tyshaieva, Alona; Damagnez, Maximilian; Strelow, Daniel; Duplessis, Ashley; Nicolai, Jessica; Wienemann, Tobias; Tamayo, Teresa; Vasconcelos, Malte Kohns; Hülse, Lisanna; Hoffmann, Katrin; Lübke, Nadine; Hauka, Sandra; Andree, Marcel; Däumer, Martin P; Thielen, Alexander; Kolbe-Busch, Susanne; Göbels, Klaus; Zotz, Rainer; Pfeffer, Klaus; Timm, Jörg; Dilthey, Alexander T; (DeCOI), Deutsche COVID-19 OMICS Initiative
In: Clin Infect Dis, pp. ciab588, 2021.
Abstract | Links | BibTeX | Tags: coronavirus, evolution, nanopore, viruses
@article{Walker:21,
title = {Characterization of SARS-CoV-2 infection clusters based on integrated genomic surveillance, outbreak analysis and contact tracing in an urban setting},
author = {Andreas Walker and Torsten Houwaart and Patrick Finzer and Lutz Ehlkes and Alona Tyshaieva and Maximilian Damagnez and Daniel Strelow and Ashley Duplessis and Jessica Nicolai and Tobias Wienemann and Teresa Tamayo and Malte Kohns Vasconcelos and Lisanna Hülse and Katrin Hoffmann and Nadine Lübke and Sandra Hauka and Marcel Andree and Martin P Däumer and Alexander Thielen and Susanne Kolbe-Busch and Klaus Göbels and Rainer Zotz and Klaus Pfeffer and Jörg Timm and Alexander T Dilthey and Deutsche COVID-19 OMICS Initiative (DeCOI)},
doi = {10.1093/cid/ciab588},
year = {2021},
date = {2021-06-28},
urldate = {2021-06-28},
journal = {Clin Infect Dis},
pages = {ciab588},
publisher = {Oxford University Press (OUP)},
abstract = {Background: Tracing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission chains is still a major challenge for public health authorities, when incidental contacts are not recalled or are not perceived as potential risk contacts. Viral sequencing can address key questions about SARS-CoV-2 evolution and may support reconstruction of viral transmission networks by integration of molecular epidemiology into classical contact tracing.
Methods: In collaboration with local public health authorities, we set up an integrated system of genomic surveillance in an urban setting, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) integration of public health authority contact tracing data, and d) a user-friendly dashboard application as a central data analysis platform.
Results: Application of the integrated system from August to December 2020 enabled a characterization of viral population structure, analysis of 4 outbreaks at a maximum care hospital, and genetically based identification of 5 putative population infection clusters, all of which were confirmed by contact tracing. The system contributed to the development of improved hospital infection control and prevention measures and enabled the identification of previously unrecognized transmission chains, involving a martial arts gym and establishing a link between the hospital to the local population.
Conclusions: Integrated systems of genomic surveillance could contribute to the monitoring and, potentially, improved management of SARS-CoV-2 transmission in the population.},
keywords = {coronavirus, evolution, nanopore, viruses},
pubstate = {published},
tppubtype = {article}
}
Background: Tracing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission chains is still a major challenge for public health authorities, when incidental contacts are not recalled or are not perceived as potential risk contacts. Viral sequencing can address key questions about SARS-CoV-2 evolution and may support reconstruction of viral transmission networks by integration of molecular epidemiology into classical contact tracing.
Methods: In collaboration with local public health authorities, we set up an integrated system of genomic surveillance in an urban setting, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) integration of public health authority contact tracing data, and d) a user-friendly dashboard application as a central data analysis platform.
Results: Application of the integrated system from August to December 2020 enabled a characterization of viral population structure, analysis of 4 outbreaks at a maximum care hospital, and genetically based identification of 5 putative population infection clusters, all of which were confirmed by contact tracing. The system contributed to the development of improved hospital infection control and prevention measures and enabled the identification of previously unrecognized transmission chains, involving a martial arts gym and establishing a link between the hospital to the local population.
Conclusions: Integrated systems of genomic surveillance could contribute to the monitoring and, potentially, improved management of SARS-CoV-2 transmission in the population.
Methods: In collaboration with local public health authorities, we set up an integrated system of genomic surveillance in an urban setting, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) integration of public health authority contact tracing data, and d) a user-friendly dashboard application as a central data analysis platform.
Results: Application of the integrated system from August to December 2020 enabled a characterization of viral population structure, analysis of 4 outbreaks at a maximum care hospital, and genetically based identification of 5 putative population infection clusters, all of which were confirmed by contact tracing. The system contributed to the development of improved hospital infection control and prevention measures and enabled the identification of previously unrecognized transmission chains, involving a martial arts gym and establishing a link between the hospital to the local population.
Conclusions: Integrated systems of genomic surveillance could contribute to the monitoring and, potentially, improved management of SARS-CoV-2 transmission in the population.
Krautwurst, Sebastian; Dijkman, Ronald; Thiel, Volker; Krumbholz, Andi; Marz, Manja
Direct RNA Sequencing for Complete Viral Genomes Book Section
In: Frishman, Dmitrij; Marz, Manja (Ed.): Virus Bioinformatics, CRC Press, 2021.
Abstract | Links | BibTeX | Tags: assembly, DNA / genomics, nanopore, nucleic acid modifications, RNA / transcriptomics, viruses
@incollection{Krautwurst:21,
title = {Direct RNA Sequencing for Complete Viral Genomes},
author = {Sebastian Krautwurst and Ronald Dijkman and Volker Thiel and Andi Krumbholz and Manja Marz},
editor = {Dmitrij Frishman and Manja Marz},
url = {https://www.taylorfrancis.com/chapters/edit/10.1201/9781003097679-3/direct-rna-sequencing-complete-viral-genomes-sebastian-krautwurst-ronald-dijkman-volker-thiel-andi-krumbholz-manja-marz},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
booktitle = {Virus Bioinformatics},
publisher = {CRC Press},
abstract = {Determination of nucleotide sequences present in biological samples (termed “sequencing”) has become a key method in almost all fields of bioscience, including virology. Since the advent of high-throughput sequencing (“second-generation sequencing”), it is possible to sequence millions of DNA fragments (“reads”) in parallel at very high accuracy, enabling the inference of single nucleotide polymorphisms (SNPs) between virus strains.
In this chapter, we provide details on how the long-read sequencing technologies (“third-generation sequencing”) which were developed in recent years have expanded the toolkit for researchers beyond the possibilities of short-read sequencing, with a focus on virus sequencing. With increased read lengths, it is possible to sequence full viral transcripts and genomes in single contiguous reads, enabling detailed studies of transcript isoforms, haplotypes, and viral quasispecies. In comparison, long-read technologies have generally higher raw read error rates, but an accurate assembly of transcripts and genomes is facilitated or made unnecessary due to the long contiguous sequences. One of the technologies, namely nanopore sequencing, also uniquely allows for direct RNA sequencing without the need for the creation or amplification of complementary DNA. This enables accurate capture of RNA content in a sample “as is,” e.g., in cells infected by RNA viruses. The protocol also leaves RNA modifications intact, which can be inferred during sequencing. Nanopore sequencing can be implemented at low costs and with constant genome coverage using cDNA amplicon sequencing methods, e.g., for highly parallel screening during virus outbreaks.},
keywords = {assembly, DNA / genomics, nanopore, nucleic acid modifications, RNA / transcriptomics, viruses},
pubstate = {published},
tppubtype = {incollection}
}
Determination of nucleotide sequences present in biological samples (termed “sequencing”) has become a key method in almost all fields of bioscience, including virology. Since the advent of high-throughput sequencing (“second-generation sequencing”), it is possible to sequence millions of DNA fragments (“reads”) in parallel at very high accuracy, enabling the inference of single nucleotide polymorphisms (SNPs) between virus strains.
In this chapter, we provide details on how the long-read sequencing technologies (“third-generation sequencing”) which were developed in recent years have expanded the toolkit for researchers beyond the possibilities of short-read sequencing, with a focus on virus sequencing. With increased read lengths, it is possible to sequence full viral transcripts and genomes in single contiguous reads, enabling detailed studies of transcript isoforms, haplotypes, and viral quasispecies. In comparison, long-read technologies have generally higher raw read error rates, but an accurate assembly of transcripts and genomes is facilitated or made unnecessary due to the long contiguous sequences. One of the technologies, namely nanopore sequencing, also uniquely allows for direct RNA sequencing without the need for the creation or amplification of complementary DNA. This enables accurate capture of RNA content in a sample “as is,” e.g., in cells infected by RNA viruses. The protocol also leaves RNA modifications intact, which can be inferred during sequencing. Nanopore sequencing can be implemented at low costs and with constant genome coverage using cDNA amplicon sequencing methods, e.g., for highly parallel screening during virus outbreaks.
In this chapter, we provide details on how the long-read sequencing technologies (“third-generation sequencing”) which were developed in recent years have expanded the toolkit for researchers beyond the possibilities of short-read sequencing, with a focus on virus sequencing. With increased read lengths, it is possible to sequence full viral transcripts and genomes in single contiguous reads, enabling detailed studies of transcript isoforms, haplotypes, and viral quasispecies. In comparison, long-read technologies have generally higher raw read error rates, but an accurate assembly of transcripts and genomes is facilitated or made unnecessary due to the long contiguous sequences. One of the technologies, namely nanopore sequencing, also uniquely allows for direct RNA sequencing without the need for the creation or amplification of complementary DNA. This enables accurate capture of RNA content in a sample “as is,” e.g., in cells infected by RNA viruses. The protocol also leaves RNA modifications intact, which can be inferred during sequencing. Nanopore sequencing can be implemented at low costs and with constant genome coverage using cDNA amplicon sequencing methods, e.g., for highly parallel screening during virus outbreaks.
2020
Overholt, Will A.; Hölzer, Martin; Geesink, Patricia; Diezel, Celia; Marz, Manja; Küsel, Kirsten
Inclusion of Oxford Nanopore long reads improves all microbial and viral metagenome-assembled genomes from a complex aquifer system Journal Article
In: Environ Microbiol, vol. 22, no. 9, pp. 4000-4013, 2020.
Abstract | Links | BibTeX | Tags: assembly, DNA / genomics, groundwater, metagenomics, nanopore, viruses
@article{Overholt:20,
title = {Inclusion of Oxford Nanopore long reads improves all microbial and viral metagenome-assembled genomes from a complex aquifer system},
author = {Will A. Overholt and Martin Hölzer and Patricia Geesink and Celia Diezel and Manja Marz and Kirsten Küsel},
doi = {10.1111/1462-2920.15186},
year = {2020},
date = {2020-08-05},
urldate = {2020-08-05},
journal = {Environ Microbiol},
volume = {22},
number = {9},
pages = {4000-4013},
publisher = {Wiley},
abstract = {Assembling microbial and viral genomes from metagenomes is a powerful and appealing method to understand structure–function relationships in complex environments. To compare the recovery of genomes from microorganisms and their viruses from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore Technologies (ONT) sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from an Illumina-only assembly, ONT-only assembly, and a hybrid assembly approach. The hybrid approach recovered 2× more mid to high-quality MAGs compared to the Illumina-only approach and 4× more than the ONT-only approach. A similar number of viral genomes were reconstructed using the hybrid and ONT methods, and both recovered nearly fourfold more viral genomes than the Illumina-only approach. While yielding fewer MAGs, the ONT-only approach generated MAGs with a high probability of containing rRNA genes, 3× higher than either of the other methods. Of the shared MAGs recovered from each method, the ONT-only approach generated the longest and least fragmented MAGs, while the hybrid approach yielded the most complete. This work provides quantitative data to inform a cost–benefit analysis of the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.},
keywords = {assembly, DNA / genomics, groundwater, metagenomics, nanopore, viruses},
pubstate = {published},
tppubtype = {article}
}
Assembling microbial and viral genomes from metagenomes is a powerful and appealing method to understand structure–function relationships in complex environments. To compare the recovery of genomes from microorganisms and their viruses from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore Technologies (ONT) sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from an Illumina-only assembly, ONT-only assembly, and a hybrid assembly approach. The hybrid approach recovered 2× more mid to high-quality MAGs compared to the Illumina-only approach and 4× more than the ONT-only approach. A similar number of viral genomes were reconstructed using the hybrid and ONT methods, and both recovered nearly fourfold more viral genomes than the Illumina-only approach. While yielding fewer MAGs, the ONT-only approach generated MAGs with a high probability of containing rRNA genes, 3× higher than either of the other methods. Of the shared MAGs recovered from each method, the ONT-only approach generated the longest and least fragmented MAGs, while the hybrid approach yielded the most complete. This work provides quantitative data to inform a cost–benefit analysis of the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.
2019
Viehweger, Adrian; Krautwurst, Sebastian; Lamkiewicz, Kevin; Madhugiri, Ramakanth; Ziebuhr, John; Hölzer, Martin; Marz, Manja
In: Genome Res, vol. 29, pp. 1545-1554, 2019.
Abstract | Links | BibTeX | Tags: assembly, coronavirus, nanopore, nucleic acid modifications, RNA / transcriptomics, viruses
@article{Viehweger:19a,
title = {Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis.},
author = {Adrian Viehweger and Sebastian Krautwurst and Kevin Lamkiewicz and Ramakanth Madhugiri and John Ziebuhr and Martin Hölzer and Manja Marz},
doi = {10.1101/gr.247064.118},
year = {2019},
date = {2019-08-22},
urldate = {2019-08-22},
journal = {Genome Res},
volume = {29},
pages = {1545-1554},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called “quasispecies.” Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.},
keywords = {assembly, coronavirus, nanopore, nucleic acid modifications, RNA / transcriptomics, viruses},
pubstate = {published},
tppubtype = {article}
}
Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called “quasispecies.” Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.