scholarly journals Identifying transposon insertions in bacterial genomes through nanopore sequencing

2019 ◽  
Author(s):  
David A. Baltrus ◽  
John Medlen ◽  
Meara Clark
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Genetic Screens ◽  
Bacterial Genomes ◽  
Sequence Capture ◽  
Transposon Insertion ◽  
Forward Genetic Screens ◽  
Transposon Insertions

AbstractTransposon mutagenesis is a widely used tool for carrying out forward genetic screens across systems, but in some cases it can be difficult to identify transposon insertion points after successful phenotypic screens. As an alternative to traditional methods, we report on the efficacy of using an Oxford Nanopore’s MinION to identify transposon insertions through whole genome sequencing. We also report experiments using CRISPR-Cas to selectively target regions of the genome where a transposon has integrated. Our experiments provide a framework for understanding the efficiency of such techniques for carrying out forward genetic screens and point towards the ability to use CRISPR-based sequence capture to identify the insertion of particular regions of DNA across all genomes, which may enable Tn-Seq experiments using Nanopore based sequencing.

scholarly journals Multiplex PCR-Based Nanopore Sequencing and Epidemiological Surveillance of Hantaan orthohantavirus in Apodemus agrarius, Republic of Korea

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 847
Author(s):  
Kyungmin Park ◽  
Seung-Ho Lee ◽  
Jongwoo Kim ◽  
Jingyeong Lee ◽  
Geum-Young Lee ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Republic Of Korea ◽  
Hantaan Virus ◽  
Genomic Sequences ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Apodemus Agrarius ◽  
Copy Numbers ◽  
Nanopore Sequencer

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

scholarly journals Real time, field-deployable whole genome sequencing of malaria parasites using nanopore technology

2020 ◽  
Author(s):  
Zahra Razook ◽  
Somya Mehra ◽  
Brittany Gilchrist ◽  
Digjaya Utama ◽  
Dulcie Lautu-Gumal ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Error Rates ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Virulence Determinants ◽  
Malaria Parasites ◽  
Additional Information ◽  
Natural Isolates

ABSTRACTMalaria parasite genomes have been generated predominantly using short read sequencing technology which can be slow, requires advanced laboratory training and does not adequately interrogate complex genomic regions that harbour important malaria virulence determinants. The portable Oxford Nanopore Technologies MinION platform generates long reads in real time and may overcome these limitations. We present compelling evidence that Nanopore sequencing delivers valuable additional information for malaria parasites with comparable data fidelity for single nucleotide variant (SNV) calls, compared to standard Illumina whole genome sequencing. We demonstrate this through sequencing of pure Plasmodium falciparum DNA, mock infections and natural isolates. Nanopore has low error rates for haploid SNV genotyping and identifies structural variants (SVs) not detected with short reads. Nanopore genomes are directly comparable to publically available genomes and produce high quality end to end chromosome assemblies. Nanopore sequencing will expedite genomic surveillance of malaria and provide new insights into parasite genome biology.

scholarly journals Forward genetic screens identified mutants with defects in trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora

2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Tsung-Yu Huang ◽  
Yi-Yun Lee ◽  
Guillermo Vidal-Diez de Ulzurrun ◽  
Yen-Ping Hsueh
Keyword(s):  
Genome Sequencing ◽  
Genetic Screening ◽  
Molecular Mechanisms ◽  
Fungal Species ◽  
Screening Methods ◽  
Whole Genome ◽  
Genetic Screens ◽  
Arthrobotrys Oligospora ◽  
Forward Genetic Screens ◽  
Functional Analyses

Abstract Nematode-trapping fungi (NTF) are carnivorous fungi that prey on nematodes under nutrient-poor conditions via specialized hyphae that function as traps. The molecular mechanisms involved in the interactions between NTF and their nematode prey are largely unknown. In this study, we conducted forward genetic screens to identify potential genes and pathways that are involved in trap morphogenesis and predation in the NTF Arthrobotrys oligospora. Using Ethyl methanesulfonate and UV as the mutagens, we generated 5552 randomly mutagenized A. oligospora strains and identified 15 mutants with strong defects in trap morphogenesis. Whole-genome sequencing and bioinformatic analyses revealed mutations in genes with roles in signaling, transcription or membrane transport that may contribute to the defects of trap morphogenesis in these mutants. We further conducted functional analyses on a candidate gene, YBP-1, and demonstrate that mutation of that gene was causative of the phenotypes observed in one of the mutants. The methods established in this study might provide helpful insights for establishing forward genetic screening methods for other non-model fungal species.

scholarly journals Mapping challenging mutations by whole-genome sequencing

2016 ◽  
Author(s):  
Harold E. Smith ◽  
Amy S. Fabritius ◽  
Aimee Jaramillo-Lambert ◽  
Andy Golden
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Single Gene ◽  
Global Scale ◽  
Model Organisms ◽  
Whole Genome ◽  
Genetic Screens ◽  
Alternative Approach ◽  
Mutation Identification

ABSTRACTWhole-genome sequencing provides a rapid and powerful method for identifying mutations on a global scale, and has spurred a renewed enthusiasm for classical genetic screens in model organisms. The most commonly characterized category of mutation consists of monogenic, recessive traits, due to their genetic tractability. Therefore, most of the mapping methods for mutation identification by whole-genome sequencing are directed toward alleles that fulfill those criteria (i.e., single-gene, homozygous variants). However, such approaches are not entirely suitable for the characterization of a variety of more challenging mutations, such as dominant and semi-dominant alleles or multigenic traits. Therefore, we have developed strategies for the identification of those classes of mutations, using polymorphism mapping in Caenorhabditis elegans as our model for validation. We also report an alternative approach for mutation identification from traditional recombinant crosses, and a solution to the technical challenge of sequencing sterile or terminally arrested strains where population size is limiting. The methods described herein extend the applicability of whole-genome sequencing to a broader spectrum of mutations, including classes that are difficult to map by traditional means.

scholarly journals Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

2020 ◽  
Vol 11 ◽  
Author(s):  
Frederico M. Batista ◽  
Tina Stapleton ◽  
James A. Lowther ◽  
Vera G. Fonseca ◽  
Rebecca Shaw ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Single Molecule ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Whole Genome ◽  
Nanopore Sequencing

scholarly journals Complete assembly of novel environmental bacterial genomes by MinIONTM sequencing

2015 ◽  
Author(s):  
Daniel J Turner ◽  
Xiaoguang Dai ◽  
Simon Mayes ◽  
Sissel Juul
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genomic Analysis ◽  
Whole Genome ◽  
Bacterial Isolates ◽  
Bacterial Genomes ◽  
Prokaryotic Genomes ◽  
Sequencing Process

In this study, we adapt a protocol for the growth of previously uncultured environmental bacterial isolates, to make it compatible with whole genome sequencing. We demonstrate that in combination with the MinION sequencing device, complete assemblies can be derived, allowing genomic comparisons to be made. This approach allows rapid, inexpensive and straightforward discovery, and genomic analysis, of previously uncultured prokaryotic genomes, and brings greater ownership of all parts of the sequencing process back to individual researchers.

scholarly journals Adaptation of Oxford Nanopore technology for hepatitis C whole genome sequencing and identification of within-host viral variants

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Nasir Riaz ◽  
Preston Leung ◽  
Kirston Barton ◽  
Martin A. Smith ◽  
Shaun Carswell ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Hepatitis C ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Reference Sequence ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Low Frequencies ◽  
Oxford Nanopore

Abstract Background Hepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here ‘nanopore sequencing’) has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing. Results The Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 samples from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per sample with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted. Conclusion Cost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.

scholarly journals Forward genetic screens identified mutants with defects in trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora

2020 ◽  
Author(s):  
Tsung-Yu Huang ◽  
Yi-Yun Lee ◽  
Guillermo Vidal-Diez de Ulzurrun ◽  
Yen-Ping Hsueh
Keyword(s):  
Genome Sequencing ◽  
Genetic Screening ◽  
Molecular Mechanisms ◽  
Fungal Species ◽  
Screening Methods ◽  
Whole Genome ◽  
Genetic Screens ◽  
Arthrobotrys Oligospora ◽  
Forward Genetic Screens ◽  
Functional Analyses

AbstractNematode-trapping fungi (NTF) are carnivorous fungi that prey on nematodes under nutrient-poor conditions via specialized hyphae that function as traps. The molecular mechanisms involved in the interactions between nematode-trapping fungi and their nematode prey are largely unknown. In this study, we conducted forward genetic screens to identify potential genes and pathways that are involved in trap morphogenesis and predation in the NTF Arthrobotrys oligospora. Using Ethyl methanesulfonate and UV as the mutagens, we generated 5552 randomly-mutagenized A. oligospora strains and identified 15 mutants with strong defects in trap morphogenesis. Whole genome sequencing and bioinformatic analyses revealed mutations in genes with roles in signaling, transcription or membrane transport that may contribute to the defects of trap morphogenesis in these mutants. We further conducted functional analyses on a candidate gene, YBP-1, and demonstrate that mutation of that gene was causative of the phenotypes observed in one of the mutants. The methods established in this study might provide helpful insights for establishing forward genetic screening methods for other non-model fungal species.

scholarly journals Integrative utility of long read sequencing-based whole genome analysis and phenotypic assay on differentiating isoniazid-resistant signature of Mycobacterium tuberculosis

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Ming-Chih Yu ◽  
Ching-Sheng Hung ◽  
Chun-Kai Huang ◽  
Cheng-Hui Wang ◽  
Yu-Chih Liang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Susceptibility Test ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Drug Resistant ◽  
Clinical Scenarios ◽  
Wide Range ◽  
Long Read

Abstract Background With the advancement of next generation sequencing technologies (NGS), whole-genome sequencing (WGS) has been deployed to a wide range of clinical scenarios. Rapid and accurate classification of drug-resistant Mycobacterium tuberculosis (MTB) would be advantageous in reducing the amplification of additional drug resistance and disease transmission. Methods In this study, a long-read sequencing approach was subjected to the whole-genome sequencing of clinical MTB clones with susceptibility test profiles, including isoniazid (INH) susceptible clones (n = 10) and INH resistant clones (n = 42) isolated from clinical specimens. Non-synonymous variants within the katG or inhA gene associated with INH resistance was identified using Nanopore sequencing coupled with a corresponding analytical workflow. Results In total, 54 nucleotide variants within the katG gene and 39 variants within the inhA gene associated with INH resistance were identified. Consistency among the results of genotypic profiles, susceptibility test, and minimal inhibitory concentration, the high-INH resistance signature was estimated using the area under the receiver operating characteristic curve with the existence of Ser315Thr (AUC = 0.822) or Thr579Asn (AUC = 0.875). Conclusions Taken together, we curated lists of coding variants associated with differential INH resistance using Nanopore sequencing, which may constitute an emerging platform for rapid and accurate identification of drug-resistant MTB clones.

scholarly journals plasmidSPAdes: Assembling Plasmids from Whole Genome Sequencing Data

2016 ◽  
Author(s):  
Dmitry Antipov ◽  
Nolan Hartwick ◽  
Max Shen ◽  
Mikhail Raiko ◽  
Alla Lapidus ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Software Tool ◽  
Software Tools ◽  
Whole Genome Sequencing Data ◽  
Antibiotics Resistance ◽  
Whole Genome ◽  
Sequencing Data ◽  
Bacterial Genomes ◽  
Specialized Software

ABSTRACTMotivationPlasmids are stably maintained extra-chromosomal genetic elements that replicate independently from the host cell’s chromosomes. Although plasmids harbor biomedically important genes, (such as genes involved in virulence and antibiotics resistance), there is a shortage of specialized software tools for extracting and assembling plasmid data from whole genome sequencing projects.ResultsWe present the plasmidSPAdes algorithm and software tool for assembling plasmids from whole genome sequencing data and benchmark its performance on a diverse set of bacterial genomes.Availability and implementationPLASMIDSPADESis publicly available athttp://spades.bioinf.spbau.ru/plasmidSPAdes/[email protected]

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