Complete Genome Sequences of Four Isolates of Vancomycin-Resistant Enterococcus faecium with the
            <i>vanA</i>
            Gene and Two Daptomycin Resistance Mutations, Obtained from Two Inpatients with Prolonged Bacteremia

Jenjaroenpun, Piroon; Wongsurawat, Thidathip; Udaondo, Zulema; Anderson, Courtney; Lopez, James; Mohan, Meera; Tytarenko, Ruslana G.; Walker, Brian A; Nookaew, Intawat; Ussery, David W.; Kothari, Atul; Jun, Se‐Ran

doi:10.1128/mra.01380-19

Cited by 6 publications

(6 citation statements)

References 14 publications

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“…The S. turicensis genomic DNA (5-day culture) was extracted with the QIAamp PowerFecal Pro DNA kit (Qiagen) and sequenced using Oxford Nanopore Technologies (ONT) and Illumina platforms to obtain a complete genome sequence of the S. turicensis isolate. The quality control and de novo assembly steps for the S. turicensis isolate were modified from reference 5 . For ONT sequencing, library preparation was done using the rapid barcoding sequencing kit (SQK-RBK004) without DNA size selection prior to sequencing with a flow cell (vR9.4/FLO-MIN106; ONT) using the MinION Mk1B sequencer (ONT) for 48 h. The raw signals from the sequencer were base called and demultiplexed using Guppy v3.2.4, followed by additional adapter trimming using Porechop v0.2.4 ( https://github.com/rrwick/Porechop ).…”

Section: Announcementmentioning

confidence: 99%

Complete Genome Sequence of Schaalia turicensis Strain CT001, Isolated from a Patient with Gonococcal Urethritis in Thailand

Nokchan

Wongsurawat

Jenjaroenpun

et al. 2021

Microbiol Resour Announc

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Schaalia turicensis , a Gram-positive bacillus, is a potential pathogen in genital infections. Here, we report the complete genome sequence of S. turicensis strain CT001, which was coisolated with Neisseria gonorrhoeae . Comprehensive analysis revealed the presence of a composite transposon carrying an imperfect class 1 integron in S. turicensis .

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Section: Announcementmentioning

confidence: 99%

Complete Genome Sequence of Schaalia turicensis Strain CT001, Isolated from a Patient with Gonococcal Urethritis in Thailand

Nokchan

Wongsurawat

Jenjaroenpun

et al. 2021

Microbiol Resour Announc

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“…ONT raw signals were base called and demultiplexed using Guppy v3.0.3 ( https://community.nanoporetech.com ). The ONT reads were then trimmed using Porechop ( https://github.com/rrwick/Porechop ), filtered using a mean quality score of 9 and a minimum read length of 2000 bases following the criteria described in [ 19 ]. The adapters and low-quality Illumina paired-end reads were trimmed using Fastp v0.19.5 [ 20 ].…”

Section: Methodsmentioning

confidence: 99%

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods

et al. 2021

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As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

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“…This MinIONmainly based method usually calibrated by NGS was used to assemble sequencing reads into genomes or plasmids, and then annotated the complete assemblies compared with resistance genes databases. Until now, AMR genes or resistance islands in drug-resistant bacterial isolates, including B. fragilis, [89] K. pneumonia, [88,92,93] A. baumannii, [94] E. coli, [72,90,[95][96][97][98] E. faecium, [99,100] N. gonorrhoeae, [101] P. aeruginosa, [102,103] B. contaminans, [104] MRSA [105] and S. suis [106] were successfully analyzed by this hybrid assembly method with an accuracy of approximate 99%. In addition, the evolution and transfer of resistance genes could also be tracked by nanopore sequencing during a prolonged infection in clinic.…”

Section: Characterization Of Antimicrobial Resistancementioning

confidence: 99%

Nanopore Electrochemistry for Pathogen Detection

Sun

et al. 2022

Chemistry An Asian Journal

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Pathogen infections seriously threaten human health, and there is an urgent demand for rapid and efficient pathogen identification to provide instructions in clinical diagnosis and therapeutic intervention. Recently, nanopore technology, a rapidly maturing technology which delivers ultrasensitive sensing and high throughput in real-time and at low cost, has achieved success in pathogen detection. Furthermore, the remarkable development of nanopore sequencing, for example, the MinION sequencer from Oxford Nanopore Technologies (ONT) as a competitive sequencing technology, has facilitated the rapid analysis of diseaserelated microbiomes at the whole-genome level and on a large scale. Here, we highlighted recent advances in nanopore approaches for pathogen detection at the singlemolecule level. We also overviewed the applications of nanopore sequencing in pathogenic bacteria identification and diagnosis. In the end, we discussed the challenges and future developments of nanopore technology as promising tools for the management of infections, which may be helpful to aid understanding as well as decision-making.

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Complete Genome Sequences of Four Isolates of Vancomycin-Resistant Enterococcus faecium with the vanA Gene and Two Daptomycin Resistance Mutations, Obtained from Two Inpatients with Prolonged Bacteremia

Cited by 6 publications

References 14 publications

Complete Genome Sequence of Schaalia turicensis Strain CT001, Isolated from a Patient with Gonococcal Urethritis in Thailand

Complete Genome Sequence of Schaalia turicensis Strain CT001, Isolated from a Patient with Gonococcal Urethritis in Thailand

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods

Nanopore Electrochemistry for Pathogen Detection

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