Rapid, multiplexed, whole genome and plasmid sequencing of foodborne pathogens using long-read nanopore technology

Abstract U.S. public health agencies have employed next-generation sequencing (NGS) as a tool to quickly identify foodborne pathogens during outbreaks. Although established short-read NGS technologies are known to provide highly accurate data, long-read sequencing is still needed to resolve highly-repetitive genomic regions and genomic arrangement, and to close the sequences of bacterial chromosomes and plasmids. Here, we report the use of long-read nanopore sequencing to simultaneously sequence the entire chromosome and plasmid of Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. We developed a rapid and random sequencing approach coupled with de novo genome assembly within a customized data analysis workflow that uses publicly-available tools. In sequencing runs as short as four hours, using the MinION instrument, we obtained full-length genomes with an average identity of 99.87% for Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These nanopore-only assemblies provided readily available information on serotype, virulence factors, and antimicrobial resistance genes. We also demonstrate the potential of nanopore sequencing assemblies for rapid preliminary phylogenetic inference. Nanopore sequencing provides additional advantages as very low capital investment and footprint, and shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies.

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Rapid, multiplexed, whole genome and plasmid sequencing of foodborne pathogens using long-read nanopore technology

10.1101/558718 ◽

2019 ◽

Cited By ~ 2

Author(s):

Tonya L. Taylor ◽

Jeremy D. Volkening ◽

Eric DeJesus ◽

Mustafa Simmons ◽

Kiril M. Dimitrov ◽

...

Keyword(s):

Foodborne Pathogens ◽

De Novo ◽

United States Public Health ◽

Turnaround Time ◽

Public Health Agencies ◽

Antimicrobial Resistance Genes ◽

Analysis Workflow ◽

Long Read ◽

Genomic Regions ◽

Next Generation Sequencing Ngs

AbstractUnited States public health agencies are focusing on next-generation sequencing (NGS) to quickly identify and characterize foodborne pathogens. Here, the MinION nanopore, long-read sequencer was used to simultaneously sequence the entire chromosome and plasmids of Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. A rapid, random sequencing approach, coupled with de novo genome assembly within a customized data analysis workflow, that can resolve highly-repetitive genomic regions, was developed. In sequencing runs, as short as four hours, using nanopore data alone, full-length genomes were obtained with an average identity of 99.87% for Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These long-read assemblies provided information on serotype, virulence factors, and antimicrobial resistance genes. Using a custom-developed, SNP-selection workflow, the potential of the nanopore-only assemblies (after only 30 minutes of sequencing) for rapid phylogenetic inference, with identical topology compared to the published dataset, was demonstrated. To achieve maximum quality assemblies, the developed bioinformatics workflow employed additional polishing steps to correct the systematic errors produced by the nanopore-only assemblies. Nanopore sequencing provided a shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies.

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Deeplasmid: Deep learning accurately separates plasmids from bacterial chromosomes

10.1101/2021.03.11.434936 ◽

2021 ◽

Author(s):

William B Andreopoulos ◽

Alexander M Geller ◽

Miriam Lucke ◽

Jan Balewski ◽

Alicia Clum ◽

...

Keyword(s):

Deep Learning ◽

High Reliability ◽

Biological Data ◽

Yersinia Ruckeri ◽

Microbial Genomes ◽

Sequencing Platform ◽

Assembly Algorithm ◽

Antimicrobial Resistance Genes ◽

Bacterial Chromosomes ◽

Long Read

AbstractPlasmids are mobile genetic elements that play a key role in microbial ecology and evolution by mediating horizontal transfer of important genes, such as antimicrobial resistance genes. Many microbial genomes have been sequenced by short read sequencers and have resulted in a mix of contigs that derive from plasmids or chromosomes. New tools that accurately identify plasmids are needed to elucidate new plasmid-borne genes of high biological importance. We have developed Deeplasmid, a deep learning tool for distinguishing plasmids from bacterial chromosomes based on the DNA sequence and its encoded biological data. It requires as input only assembled sequences generated by any sequencing platform and assembly algorithm and its runtime scales linearly with the number of assembled sequences. Deeplasmid achieves an AUC-ROC of over 93%, and it was much more precise than the state-of-the-art methods. Finally, as a proof of concept, we used Deeplasmid to predict new plasmids in the fish pathogen Yersinia ruckeri ATCC 29473 that has no annotated plasmids. Deeplasmid predicted with high reliability that a long assembled contig is part of a plasmid. Using long read sequencing we indeed validated the existence of a 102 Kbp long plasmid, demonstrating Deeplasmid’s ability to detect novel plasmids.AvailabilityThe software is available with a BSD license: deeplasmid.sourceforge.io. A Docker container is available on DockerHub under: billandreo/[email protected]@mail.huji.ac.il

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Comparative analysis of alignment tools for application on Nanopore sequencing data

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2021-2212 ◽

2021 ◽

Vol 7 (2) ◽

pp. 831-834

Author(s):

Chiara Becht ◽

Jonas Schmidt ◽

Frithjof Blessing ◽

Folker Wenzel

Keyword(s):

Error Rate ◽

De Novo ◽

Performance Criteria ◽

Computational Time ◽

Nanopore Sequencing ◽

Sequencing Data ◽

Accurate Analysis ◽

Match Rate ◽

Long Read ◽

And Performance

Abstract INTRODUCTION: Long-read sequencing techniques such as Oxford Nanopore sequencing, are representing a promising novel approach in molecular-biological methodology, enabling potential facilitation in mapping and de novo assembly. In comparison to conventional sequencing methods, novel alignment tools are mandated to compensate differing data structures (especially high error rate) to achieve acceptably accurate analysis results. METHODS: In this study, benchmarking for long read aligners BLASR, GraphMap, LAST, minimap2, NGMLR and the short-read aligner BWA MEM on three experimental datasets was conducted. Obtained alignment results were compared for various quality and performance criteria, such as match rate, mismatch rate, error rate, working memory usage and computational time. RESULTS: The comparison yielded differences in alignment quality and performance of tools under test. Tool LAST showed the largest differences among all tools. Minimap2 achieved constant quality with good performance. BLASR, GraphMap, BWA MEM and NGMLR showed slight differences only. CONCLUSION: Differences among the tools could be reasoned with dataset characteristics and algorithm approaches of individual tools. All tools except BLASR seem applicable for Nanopore sequencing data. Therefore, selection of the tool should be done under consideration of the experimental design and the further downstream analysis

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De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽

10.12688/f1000research.11146.2 ◽

2018 ◽

Vol 6 ◽

pp. 618 ◽

Cited By ~ 2

Author(s):

Michael Liem ◽

Hans J. Jansen ◽

Ron P. Dirks ◽

Christiaan V. Henkel ◽

G. Paul H. van Heusden ◽

...

Keyword(s):

De Novo ◽

Sequence Data ◽

New Technology ◽

Whole Genome ◽

Nanopore Sequencing ◽

Short Read ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Before And After ◽

Long Read

Background: The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

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De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽

10.12688/f1000research.11146.1 ◽

2017 ◽

Vol 6 ◽

pp. 618 ◽

Cited By ~ 3

Author(s):

Hans J. Jansen ◽

Ron P. Dirks ◽

Michael Liem ◽

Christiaan V. Henkel ◽

G. Paul H. van Heusden ◽

...

Keyword(s):

De Novo ◽

Sequence Data ◽

New Technology ◽

Whole Genome ◽

Nanopore Sequencing ◽

Short Read ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read ◽

Wild Type Yeast

Background: The introduction of the MinIONTM sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. It has been shown that the nanopore sequence data, in combination with other sequencing technologies, is highly useful for accurate annotation of all genes in the genome. However, it also offers great potential for de novo assembly of complex genomes without using other technologies. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

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Long-read sequencing identified a novel nonsense and a de novo missense of PPA2 in trans in a Chinese patient with autosomal recessive infantile sudden cardiac failure

Clinica Chimica Acta ◽

10.1016/j.cca.2021.03.029 ◽

2021 ◽

Author(s):

Arman Zhao ◽

Jie Shen ◽

Yueyue Ding ◽

Mao Sheng ◽

Mengying Zuo ◽

...

Keyword(s):

Cardiac Failure ◽

Autosomal Recessive ◽

De Novo ◽

Chinese Patient ◽

Long Read ◽

In Trans

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MBRS-47. RAPID MOLECULAR SUBGROUPING OF MEDULLOBLASTOMA BASED ON DNA METHYLATION BY NANOPORE SEQUENCING

Neuro-Oncology ◽

10.1093/neuonc/noaa222.556 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii406-iii406

Author(s):

Julien Masliah-Planchon ◽

Elodie Girard ◽

Philipp Euskirchen ◽

Christine Bourneix ◽

Delphine Lequin ◽

...

Keyword(s):

Dna Methylation ◽

Single Molecule ◽

Nanopore Sequencing ◽

Molecular Subgroup ◽

Group Assignment ◽

Group 4 ◽

Methylation Assay ◽

Tumor Group ◽

Long Read ◽

Group 3

Abstract Medulloblastoma (MB) can be classified into four molecular subgroups (WNT group, SHH group, group 3, and group 4). The gold standard of assignment of molecular subgroup through DNA methylation profiling uses Illumina EPIC array. However, this tool has some limitation in terms of cost and timing, in order to get the results soon enough for clinical use. We present an alternative DNA methylation assay based on nanopore sequencing efficient for rapid, cheaper, and reliable subgrouping of clinical MB samples. Low-depth whole genome with long-read single-molecule nanopore sequencing was used to simultaneously assess copy number profile and MB subgrouping based on DNA methylation. The DNA methylation data generated by Nanopore sequencing were compared to a publicly available reference cohort comprising over 2,800 brain tumors including the four subgroups of MB (Capper et al. Nature; 2018) to generate a score that estimates a confidence with a tumor group assignment. Among the 24 MB analyzed with nanopore sequencing (six WNT, nine SHH, five group 3, and four group 4), all of them were classified in the appropriate subgroup established by expression-based Nanostring subgrouping. In addition to the subgrouping, we also examine the genomic profile. Furthermore, all previously identified clinically relevant genomic rearrangements (mostly MYC and MYCN amplifications) were also detected with our assay. In conclusion, we are confirming the full reliability of nanopore sequencing as a novel rapid and cheap assay for methylation-based MB subgrouping. We now plan to implement this technology to other embryonal tumors of the central nervous system.

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Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq

Nature Biotechnology ◽

10.1038/s41587-021-00965-w ◽

2021 ◽

Author(s):

Martin Philpott ◽

Jonathan Watson ◽

Anjan Thakurta ◽

Tom Brown ◽

...

Keyword(s):

Single Cell ◽

Error Detection ◽

Single Cells ◽

Fusion Transcript ◽

Building Blocks ◽

Myeloma Cell ◽

Nanopore Sequencing ◽

Long Read ◽

Unique Molecular Identifier ◽

Transcript Detection

AbstractHere we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.

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How Long Are Long Tandem Repeats? A Challenge for Current Methods of Whole-Genome Sequence Assembly: The Case of Satellites in Caenorhabditis elegans

Genes ◽

10.3390/genes9100500 ◽

2018 ◽

Vol 9 (10) ◽

pp. 500

Author(s):

Juan A. Subirana ◽

Xavier Messeguer

Keyword(s):

Caenorhabditis Elegans ◽

Sanger Sequencing ◽

Tandem Repeats ◽

Whole Genome Sequence ◽

Nanopore Sequencing ◽

Original Sequence ◽

Genome Sequence Assembly ◽

Long Read ◽

Genomic Regions ◽

Caenorhabditis Elegans Genome

Repetitive genome regions have been difficult to sequence, mainly because of the comparatively small size of the fragments used in assembly. Satellites or tandem repeats are very abundant in nematodes and offer an excellent playground to evaluate different assembly methods. Here, we compare the structure of satellites found in three different assemblies of the Caenorhabditis elegans genome: the original sequence obtained by Sanger sequencing, an assembly based on PacBio technology, and an assembly using Nanopore sequencing reads. In general, satellites were found in equivalent genomic regions, but the new long-read methods (PacBio and Nanopore) tended to result in longer assembled satellites. Important differences exist between the assemblies resulting from the two long-read technologies, such as the sizes of long satellites. Our results also suggest that the lengths of some annotated genes with internal repeats which were assembled using Sanger sequencing are likely to be incorrect.

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