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 Whole-Genome Sequencing of a Human Clinical Isolate of the Novel Species Klebsiella quasivariicola sp. nov

2017 ◽  
Vol 5 (42) ◽  
Author(s):  
S. Wesley Long ◽  
Sarah E. Linson ◽  
Matthew Ojeda Saavedra ◽  
Concepcion Cantu ◽  
James J. Davis ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Novel Species ◽  
Whole Genome ◽  
The Novel ◽  
Short Read ◽  
Oxford Nanopore ◽  
Long Read

ABSTRACT In a study of 1,777 Klebsiella strains, we discovered KPN1705, which was distinct from all recognized Klebsiella spp. We closed the genome of strain KPN1705 using a hybrid of Illumina short-read and Oxford Nanopore long-read technologies. For this novel species, we propose the name Klebsiella quasivariicola sp. nov.

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 Mobile Colistin Resistance Genetic Determinants of Non-Typhoid Salmonella enterica Isolates from Russia

2021 ◽  
Vol 9 (12) ◽  
pp. 2515
Author(s):  
Konstantin V. Kuleshov ◽  
Anastasia S. Pavlova ◽  
Elizaveta D. Shedko ◽  
Yulia V. Mikhaylova ◽  
Gabriele Margos ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enterica ◽  
Diarrheal Disease ◽  
Whole Genome ◽  
Genetic Determinants ◽  
Colistin Resistance ◽  
Oxford Nanopore ◽  
Susceptible Isolate ◽  
Long Read

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently poses a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carrying mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n = 586). mcr-1 genes were detected using a previously developed qPCR assay, and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates, including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans, carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene-carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological backgrounds.

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 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 Complete nontuberculous mycobacteria whole genomes using an optimized DNA extraction protocol for long-read sequencing

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Jennifer M. Bouso ◽  
Paul J. Planet
Keyword(s):  
Cell Wall ◽  
Dna Extraction ◽  
Extraction Method ◽  
Nontuberculous Mycobacteria ◽  
Systemic Disease ◽  
Matrix Material ◽  
Enzymatic Digestion ◽  
Extraction Protocol ◽  
Long Read ◽  
Assembly Pipeline

Abstract Background Nontuberculous 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 provide new opportunities to sequence and construct complete bacterial genomes faster and at a lower cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to its 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 with no additional clean-up steps. Results Our DNA extraction method was compared to 6 other methods with variations in timing of mechanical disruption and enzymatic digestion of the cell wall, quantity of matrix material, and reagents used in extraction and precipitation. We tested our optimized method on 38 clinical isolates from the M. avium and M. abscessus complexes, which yielded optimal quality and quantity measurements for Oxford Nanopore Technologies sequencing. We also present the efficient completion of circularized M. avium subspecies hominissuis genomes using our extraction technique and the long-read sequencing MinION platform, including the identification of a novel plasmid. Conclusions Our optimized extraction protocol and assembly pipeline was both sufficient and efficient for genome closure. We expect that our finely-tuned extraction method will prove to be a valuable tool in long-read sequencing and completion of mycobacterial genomes going forward. Utilization of comprehensive, long-read based approaches will advance the understanding evolution and pathogenicity of NTM infections.

scholarly journals Mobile Colistin Resistance Genetic Determinants of Non-typhoid Salmonella enterica Isolates from Russia

2021 ◽  
Author(s):  
K. V. Kuleshov ◽  
A. S. Pavlova ◽  
E. D. Shedko ◽  
Y. V. Mikhaylova ◽  
G. Margos ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enterica ◽  
Diarrheal Disease ◽  
Whole Genome ◽  
Genetic Determinants ◽  
Colistin Resistance ◽  
Oxford Nanopore ◽  
Susceptible Isolate ◽  
Long Read

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently pose a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carries mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n=586). mcr-1 genes were detected using a previously developed qPCR assay and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological background.

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 A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathy E. Raven ◽  
Sophia T. Girgis ◽  
Asha Akram ◽  
Beth Blane ◽  
Danielle Leek ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
Simultaneous Processing ◽  
Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

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