scholarly journals Whole-Genome Sequencing of a Human Clinical Isolate of emm28 Streptococcus pyogenes Causing Necrotizing Fasciitis Acquired Contemporaneously with Hurricane Harvey

2017 ◽  
Vol 5 (45) ◽  
Author(s):  
S. Wesley Long ◽  
Priyanka Kachroo ◽  
James M. Musser ◽  
Randall J. Olsen
Keyword(s):  
Necrotizing Fasciitis ◽  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Streptococcus Pyogenes ◽  
Metropolitan Area ◽  
Sequence Data ◽  
Whole Genome ◽  
Content Type ◽  
Oxford Nanopore

ABSTRACT We discovered an emm28 Streptococcus pyogenes isolate causing necrotizing fasciitis in a patient exposed to the floodwaters of Hurricane Harvey in the Houston, TX, metropolitan area in August 2017. The Oxford Nanopore MinION instrument provided sufficient genome sequence data within 1 h of beginning sequencing to close the genome.

scholarly journals Whole-Genome Sequencing of Mycobacterium tilburgii Strain MEPHI

2019 ◽  
Vol 8 (40) ◽  
Author(s):  
Jamal Saad ◽  
Michel Drancourt ◽  
Margaret M. Hannan ◽  
Patrick J. Stapleton ◽  
Simon Grandjean Lapierre
Keyword(s):  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Gc Content ◽  
Whole Genome ◽  
Content Type ◽  
Phylogenetic Placement ◽  
Mycobacterium Simiae

Mycobacterium tilburgii is a fastidious mycobacterium which has previously been reported to cause severe disseminated infections. Genome sequencing of the M. tilburgii MEPHI clinical isolate yielded 3.14 Mb, with 66.3% GC content, and confirmed phylogenetic placement within the Mycobacterium simiae complex.

scholarly journals Genome Sequencing of Polydrug-, Multidrug-, and Extensively Drug-Resistant Mycobacterium tuberculosis Strains from South India

2019 ◽  
Vol 8 (12) ◽  
Author(s):  
Sivakumar Shanmugam ◽  
Narender Kumar ◽  
Dina Nair ◽  
Mohan Natrajan ◽  
Srikanth Prasad Tripathy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
South India ◽  
Sequence Data ◽  
Whole Genome ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Content Type ◽  
Extensively Drug Resistant

The genomes of 16 clinical Mycobacterium tuberculosis isolates were subjected to whole-genome sequencing to identify mutations related to resistance to one or more anti-Mycobacterium drugs. The sequence data will help in understanding the genomic characteristics of M. tuberculosis isolates and their resistance mutations prevalent in South India.

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 Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing

2015 ◽  
Vol 53 (8) ◽  
pp. 2716-2719 ◽  
Author(s):  
K. Bjorn-Mortensen ◽  
J. Zallet ◽  
T. Lillebaek ◽  
A. B. Andersen ◽  
S. Niemann ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Whole Genome ◽  
Content Type ◽  
Direct Dna Extraction ◽  
Slow Growing

Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such asMycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks.

scholarly journals Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding

2020 ◽  
Author(s):  
Nikki E. Freed ◽  
Markéta Vlková ◽  
Muhammad B. Faisal ◽  
Olin K. Silander
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Variant Calling ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Cost Efficient ◽  
Primer Sets ◽  
Library Preparation Method

AbstractRapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

scholarly journals Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Nikki E Freed ◽  
Markéta Vlková ◽  
Muhammad B Faisal ◽  
Olin K Silander
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Variant Calling ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Cost Efficient ◽  
Primer Sets ◽  
Library Preparation Method

Abstract Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

scholarly journals CTX-M-15-Producing Shewanella Species Clinical Isolate Expressing OXA-535, a Chromosome-Encoded OXA-48 Variant, Putative Progenitor of the Plasmid-Encoded OXA-436

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Agnès B. Jousset ◽  
Laura Dabos ◽  
Rémy A. Bonnin ◽  
Delphine Girlich ◽  
Anaïs Potron ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Bacterial Species ◽  
Whole Genome ◽  
Biochemical Tests ◽  
Bile Sample ◽  
Content Type ◽  
E Coli ◽  
Shewanella Species

ABSTRACT Shewanella spp. constitute a reservoir of antibiotic resistance determinants. In a bile sample, we identified three extended-spectrum-β-lactamase (ESBL)-producing bacteria (Escherichia coli, Klebsiella pneumoniae, and Shewanella sp. strain JAB-1) isolated from a child suffering from cholangitis. Our objectives were to characterize the genome and the resistome of the first ESBL-producing isolate of the genus Shewanella and determine whether plasmidic exchange occurred between the three bacterial species. Bacterial isolates were characterized using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), standard biochemical tools, and antimicrobial susceptibility testing. Shewanella sp. JAB-1 and ESBL gene-encoding plasmids were characterized using PacBio and Illumina whole-genome sequencing, respectively. The Shewanella sp. JAB-1 chromosome-encoded OXA-48 variant was cloned and functionally characterized. Whole-genome sequencing (WGS) of the Shewanella sp. clinical isolate JAB-1 revealed the presence of a 193-kb plasmid belonging to the IncA/C incompatibility group and harboring two ESBL genes, bla CTX-M-15 and bla SHV-2a. bla CTX-M-15 gene-carrying plasmids belonging to the IncY and IncR incompatibility groups were also found in the E. coli and K. pneumoniae isolates from the same patient, respectively. A comparison of the bla CTX-M-15 genetic environment indicated the independent origin of these plasmids and dismissed in vivo transfers. Furthermore, characterization of the resistome of Shewanella sp. JAB-1 revealed the presence of a chromosome-carried bla OXA-535 gene, likely the progenitor of the plasmid-carried bla OXA-436 gene, a novel bla OXA-48-like gene. The expression of bla OXA-535 in E. coli showed the carbapenem-hydrolyzing activity of OXA-535. The production of OXA-535 in Shewanella sp. JAB-1 could be evidenced using molecular and immunoenzymatic tests, but not with biochemical tests that monitor carbapenem hydrolysis. In this study, we have identified a CTX-M-15-producing Shewanella species that was responsible for a hepatobiliary infection and that is likely the progenitor of OXA-436, a novel plasmid-encoded OXA-48-like class D carbapenemase.

scholarly journals Lowering the Barriers to Routine Whole-Genome Sequencing of Bacteria in the Clinical Microbiology Laboratory

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Daniel D. Rhoads
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Sequence Data ◽  
Whole Genome Sequence ◽  
Clinical Microbiology Laboratory ◽  
Whole Genome ◽  
Content Type ◽  
Antibacterial Susceptibility ◽  
Susceptibility And Resistance

ABSTRACT Whole-genome sequencing of bacterial isolates is increasingly being used to predict antibacterial susceptibility and resistance. Mason and coauthors describe the phenotypic susceptibility interpretations of more than 1,300 Staphylococcus aureus isolates tested against a dozen antistaphylococcal agents, and they compared these findings to susceptibility predictions made by analyzing whole-genome sequence data (J Clin Microbiol 56:e01815-17, 2018, https://doi.org/10.1128/JCM.01815-17). The genotype-phenotype susceptibility interpretations correlated in 96.3% (2,720/2,825) of resistant findings and 98.8% (11,504/11,639) of susceptible findings. This work by Mason and colleagues is helping to lower the barriers to using whole-genome sequencing of S. aureus in clinical microbiology practice.

scholarly journals Isolation and Enrichment of Cryptosporidium DNA and Verification of DNA Purity for Whole-Genome Sequencing

2014 ◽  
Vol 53 (2) ◽  
pp. 641-647 ◽  
Author(s):  
Yaqiong Guo ◽  
Na Li ◽  
Colleen Lysén ◽  
Michael Frace ◽  
Kevin Tang ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genomic Dna ◽  
Sanger Sequencing ◽  
Sequence Data ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Genotype I ◽  
Content Type ◽  
Dna Purity

Whole-genome sequencing ofCryptosporidiumspp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment ofCryptosporidiumgenomic DNA from fecal specimens and verification of DNA purity for whole-genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole-genome amplification (WGA) of DNA from purified oocysts. Quantitative PCR (qPCR) analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next-generation sequencing tools were used in final evaluations of genome coverage and of the extent of contamination. Altogether, 24 fecal specimens ofCryptosporidium parvum,C. hominis,C. andersoni,C. ubiquitum,C. tyzzeri, andCryptosporidiumchipmunk genotype I were processed with the procedures. As expected, WGA products with low (<16.0) threshold cycle (CT) values yielded mostlyCryptosporidiumsequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived fromCryptosporidiumgenomic DNA, ≤25%). Following this strategy, 20 WGA products from sixCryptosporidiumspecies or genotypes with low (mostly <14.0)CTvalues were submitted to whole-genome sequencing, generating sequence data covering 94.5% to 99.7% ofCryptosporidiumgenomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment ofCryptosporidiumDNA from fecal specimens for whole-genome sequencing.

scholarly journals KPC-53, a KPC-3 Variant of Clinical Origin Associated with Reduced Susceptibility to Ceftazidime-Avibactam

2020 ◽  
Vol 65 (1) ◽  
pp. e01429-20
Author(s):  
Vincenzo Di Pilato ◽  
Noemi Aiezza ◽  
Valentina Viaggi ◽  
Alberto Antonelli ◽  
Luigi Principe ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Mechanisms Of Resistance ◽  
Content Type ◽  
High Level ◽  
Level Resistance

ABSTRACTThis study reports on the characterization of a Klebsiella pneumoniae clinical isolate showing high-level resistance to ceftazidime-avibactam associated with the production of KPC-53, a KPC-3 variant exhibiting a Leu167Glu168 duplication in the Ω-loop and a loss of carbapenemase activity. Whole-genome sequencing (WGS) revealed the presence of two copies of blaKPC-53, located on a pKpQIL-like plasmid and on a plasmid prophage of the Siphoviridae family, respectively. The present findings provide new insights into the mechanisms of resistance to ceftazidime-avibactam.

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