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

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.

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

10.1101/2020.12.17.423341 ◽

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.

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Identifying transposon insertions in bacterial genomes through nanopore sequencing

10.1101/765545 ◽

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.

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

Genes ◽

10.3390/genes11121444 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1444

Author(s):

Nazeefa Fatima ◽

Anna Petri ◽

Ulf Gyllensten ◽

Lars Feuk ◽

Adam Ameur

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Single Molecule ◽

Large Scale ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Structural Variations ◽

Single Molecule Sequencing ◽

Human Samples

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

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Linked-Read Whole Genome Sequencing Solves a Double DMD Gene Rearrangement

Genes ◽

10.3390/genes12020133 ◽

2021 ◽

Vol 12 (2) ◽

pp. 133

Author(s):

Maria Elena Onore ◽

Annalaura Torella ◽

Francesco Musacchia ◽

Paola D’Ambrosio ◽

Mariateresa Zanobio ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Single Molecule ◽

Genetic Disorders ◽

Carrier Status ◽

Female Carrier ◽

Whole Genome ◽

Her Family ◽

Dmd Gene ◽

Normal Dosage

Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16–29 in DMD gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a “non-carrier” status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1–15 and 30–34. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16–29 deletion, while the second allele showed a 1–34 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16–29 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.

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

BMC Genomics ◽

10.1186/s12864-021-07460-1 ◽

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.

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

Journal of Biomedical Science ◽

10.1186/s12929-021-00783-x ◽

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.

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