scholarly journals DNA Thermo-Protection Facilitates Whole Genome Sequencing of Mycobacteria Direct from Clinical Samples by the Nanopore Platform

2020 ◽  
Author(s):  
Sophie George ◽  
Yifei Xu ◽  
Gillian Rodger ◽  
Marcus Morgan ◽  
Nicholas D. Sanderson ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Low Cost ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Whole Genome ◽  
Genome Coverage ◽  
Human Dna ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

ABSTRACTMycobacterium tuberculosis (MTB) is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for Oxford Nanopore Technologies (ONT) sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99°C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing. Initial validation employed Mycobacteria DNA (extracted or intracellular). Next, mock clinical samples (infection-negative human sputum spiked 0-105 BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat-inactivation. DNA was extracted and sequenced. Human DNA degraded faster than Mycobacteria DNA, resulting in target enrichment. Four replicate experiments each demonstrated detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) was achieved at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealed initial sample volumes ≥1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% five-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.

scholarly journals DNA Thermo-Protection Facilitates Whole-Genome Sequencing of Mycobacteria Direct from Clinical Samples

2020 ◽  
Vol 58 (10) ◽  
Author(s):  
Sophie George ◽  
Yifei Xu ◽  
Gillian Rodger ◽  
Marcus Morgan ◽  
Nicholas D. Sanderson ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Low Cost ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Whole Genome ◽  
Genome Coverage ◽  
Content Type ◽  
C 30 ◽  
Validation Experiments

ABSTRACT Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 105 Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

scholarly journals Assessment of Metagenomic MinION and Illumina sequencing as an approach for the recovery of whole genome sequences of chikungunya and dengue viruses directly from clinical samples

2018 ◽  
Author(s):  
Liana E. Kafetzopoulou ◽  
Kyriakos Efthymiadis ◽  
Kuiama Lewandowski ◽  
Ant Crook ◽  
Dan Carter ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Clinical Samples ◽  
Whole Genome ◽  
Metagenomic Sequencing ◽  
Front Line ◽  
Genome Sequences ◽  
Oxford Nanopore

AbstractThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION to study the feasibility of whole genome sequencing from clinical samples containing chikungunya or dengue virus, two of the most important arboviruses. Direct metagenomic sequencing of nucleic acid extracts from serum and plasma without viral enrichment allowed for virus and coinfection identification, subtype determination and in the majority of cases elucidated complete or near-complete genomes adequate for phylogenetic analysis. This work demonstrates that metagenomic whole genome sequencing is feasible for over 90% and 80% of chikungunya and dengue virus PCR-positive patient samples respectively. It confirms the feasibility of field metagenomic sequencing for these and likely other RNA viruses, highlighting the applicability of this approach to front-line public health.

scholarly journals Identification and Whole-Genome Sequencing of a Monkeypox Virus Strain Isolated in Israel

2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Inbar Cohen-Gihon ◽  
Ofir Israeli ◽  
Ohad Shifman ◽  
Noam Erez ◽  
Sharon Melamed ◽  
...  
Keyword(s):  
West Africa ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Virus Strain ◽  
Illumina Miseq ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Monkeypox Virus ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

We report the whole-genome sequence of a monkeypox virus strain isolated in Israel. The strain was isolated in 2018 from a patient travelling back from West Africa. The virus was fully sequenced on the Illumina MiSeq and Oxford Nanopore Technologies MinION platforms.

scholarly journals Whole genome sequencing and antibiotic diffusion assays, provide new insight on drug resistance in the genus Pedobacter

2020 ◽  
Vol 96 (6) ◽  
Author(s):  
Ingvild Falkum Ullmann ◽  
Anders Benteson Nygaard ◽  
Hege Smith Tunsjø ◽  
Colin Charnock
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Drinking Water Treatment ◽  
Whole Genome ◽  
Sequencing Data ◽  
Multidrug Efflux ◽  
Beta Lactams ◽  
Sequencing Platform ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

ABSTRACT A total of four strains of the ‘environmental superbug’ Pedobacter isolated from sludge produced at Norwegian drinking water treatment plants, were characterized by whole genome sequencing and antibiotic susceptibility assays. As with previous studies on members of this genus, we found that the isolates were multi-drug resistant, and that this resistance included clinically important beta-lactams, aminoglycosides and the fluoroquinolone ciprofloxacin. Using the minION sequencing platform (Oxford Nanopore Technologies) combined with HiSeq PE150 Illumina sequencing data, the four isolates were assembled into genomes of single contigs. Analysis of the genomes revealed potential genetic factors possibly underlying some of the specific resistances observed. Metallo-beta-lactamase activity was detected in one isolate, and the same isolate contained a putative metallo-betalactamase gene resembling pedo-2. Furthermore, several genes related to multidrug efflux systems were found using the resistance database CARD. Additionally, the present study extends our knowledge on the phylogeny of this genus, adding four new genomes to the existing 50.

scholarly journals DNA Extraction Method Optimized for Nontuberculous Mycobacteria Long-Read Whole Genome Sequencing

2018 ◽  
Author(s):  
Jennifer M. Bouso ◽  
Paul J. Planet
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Nontuberculous Mycobacteria ◽  
Systemic Disease ◽  
Matrix Material ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies

AbstractNontuberculous mycobacteria (NTM) are a major cause of pulmonary and systemic disease in at-risk populations. Gaps in knowledge about transmission patterns, evolution, and pathogenicity during infection have prompted a recent surge in genomic NTM research. Increased availability and affordability of whole genome sequencing (WGS) techniques, including the advent of Oxford Nanopore Technologies, provide new opportunities to sequence complete NTM genomes at a fraction of the previous cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to their slow growth and recalcitrant cell wall. Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA. Our refined method was compared to 6 other methods with variations in timing of mechanical and enzymatic digestion, quantity of matrix material, and reagents used in extraction and precipitation. We also demonstrate the ability of our optimized protocol to produce sufficient DNA to yield near-complete NTM genome assemblies using Oxford Nanopore Technologies long-read sequencing.

scholarly journals Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables easy, fast and accurate outbreak analysis in hospital and community settings

2020 ◽  
Author(s):  
Femke Wolters ◽  
Jordy P.M. Coolen ◽  
Alma Tostmann ◽  
Lenneke F.J. van Groningen ◽  
Chantal P. Bleeker-Rovers ◽  
...  
Keyword(s):  
Public Health ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Healthcare Workers ◽  
Whole Genome ◽  
Sequencing Technology ◽  
Reverse Complement ◽  
Oxford Nanopore ◽  
Epidemiological Link ◽  
Oxford Nanopore Technologies

AbstractBackgroundCurrent transmission rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still increasing and many countries are facing second waves of infections. Rapid SARS-CoV-2 whole-genome sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring and determining transmission links. Here we report the use of reverse complement polymerase chain reaction (RC-PCR), a novel technology for WGS of SARS-CoV-2 enabling library preparation in a single PCR saving time, resources and enables high throughput screening. Additionally, we show SARS-CoV-2 diversity and possible transmission within the Radboud university medical center (Radboudumc) during September 2020 using RC-PCR WGS.MethodsA total of 173 samples tested positive for SARS-CoV-2 between March and September 2020 were selected for whole-genome sequencing. Ct values of the samples ranged from 16 to 42. They were collected from 83 healthcare workers and three patients at the Radboudumc, in addition to 64 people living in the area around the hospital and tested by the local health services. For validation purposes, nineteen of the included samples were previously sequenced using Oxford Nanopore Technologies and compared to RC-PCR WGS results. The applicability of RC-PCR WGS in outbreak analysis for public health service and hospitals was tested on six suspected clusters containing samples of healthcare workers and patients with an epidemiological link.FindingsRC-PCR resulted in sequencing data for 146 samples. It showed a genome coverage of up to 98,2% for samples with a maximum Ct value of 32. Comparison to Oxford Nanopore technologies gives a near-perfect agreement on 95% of the samples (18 out of 19). Three out of six clusters with a suspected epidemiological link were fully confirmed, in the others, four healthcare workers were not associated. In the public health service samples, a previously unknown chain of transmission was confirmed.Significance statementSAR-CoV-2 whole-genome sequencing using RC-PCR is a reliable technique and applicable for use in outbreak analysis and surveillance. Its ease of use, high-trough screening capacity and wide applicability makes it a valuable addition or replacement during this ongoing SARS-CoV-2 pandemic.FundingNoneResearch in contextEvidence before this studyAt present whole genome sequencing techniques for SARS-CoV-2 have a large turnover time and are not widely available. Only a few laboratories are currently able to perform large scale SARS-CoV-2 sequencing. This restricts the use of sequencing to aid hospital and community infection prevention.Added value of this studyHere we present clinical and technical data on a novel Whole Genome Sequencing technology, implementing reverse-complement PCR. It is able to obtain high genome coverage of SARS-CoV-2 and confirm and exclude epidemiological links in 173 healthcare workers and patients. The RC-PCR technology simplifies the workflow thereby reducing hands on time. It combines targeted PCR and sequence library construction in a single PCR, which normally takes several steps. Additionally, this technology can be used in concordance with the widely available range of Illumina sequencers.Implications of all the available evidenceRC-PCR whole genome sequencing technology enables rapid and targeted surveillance and response to an ongoing outbreak that has great impact on public health and society. Increased use of sequencing technologies in local laboratories can help prevent increase of SARS-CoV-2 spreading by better understanding modes of transmission.

scholarly journals Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Jacqueline King ◽  
Anne Pohlmann ◽  
Kamila Dziadek ◽  
Martin Beer ◽  
Kerstin Wernike
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Economic Losses ◽  
Clinical Samples ◽  
Bovine Viral Diarrhea ◽  
Sequence Information ◽  
Whole Genome ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Loads

Abstract Background As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5’ untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. Results To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19–32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. Conclusions Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

scholarly journals SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Weili Cai ◽  
Schyler Nunziata ◽  
John Rascoe ◽  
Michael J. Stulberg
Keyword(s):  
Whole Genome Sequencing ◽  
Molecular Characterization ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genome Coverage ◽  
Metagenomic Sample ◽  
Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

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