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Article ; Online: Draft Genome Sequence of Multidrug Resistant Klebsiella pneumoniae Strain C43 Isolated from Environmental Water Sample.

Wee, Soon Keong / Yap, Eric Peng Huat

Microbiology resource announcements

2022 Volume 11, Issue 9, Page(s) e0025222

Abstract: Here, we report the genome of ESBL-producing Klebsiella pneumoniae strain C43, which was isolated from an environmental water sample. The genome is 5,614,412 bp in size with GC content of 56.86% with multidrug antimicrobial resistance genes and several ... ...

Abstract	Here, we report the genome of ESBL-producing Klebsiella pneumoniae strain C43, which was isolated from an environmental water sample. The genome is 5,614,412 bp in size with GC content of 56.86% with multidrug antimicrobial resistance genes and several metal resistance gene operons.
Language	English
Publishing date	2022-08-22
Publishing country	United States
Document type	Journal Article
ISSN	2576-098X
ISSN (online)	2576-098X
DOI	10.1128/mra.00252-22
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Pre-isolation Molecular Screening for High-throughput Sampling and Sequencing of Bacterial Microbes from the Environment.

Wee, Soon Keong / Chan, Mark Boon Pho / Sivalingam, Suppiah Paramalingam / Yap, Eric Peng Huat

Current protocols

2023 Volume 3, Issue 5, Page(s) e778

Abstract: Environmental studies often require culture and characterization to understand the prevalence, distribution, persistence and functions of target microorganisms in ecological habitats. Isolating pure microbiological monocultures allows the phenotypic ... ...

Abstract	Environmental studies often require culture and characterization to understand the prevalence, distribution, persistence and functions of target microorganisms in ecological habitats. Isolating pure microbiological monocultures allows the phenotypic characterization of microorganisms to study their functional properties. For efficient isolation of low-prevalence organisms, enrichment followed by PCR screening is performed to identify positive samples for subsequent culture. Molecular characterization, strain-typing, and genotyping of isolated microorganisms is best comprehensively performed using whole-genome sequencing. This article outlines end-to-end protocols for screening, isolation, and sequencing of microbes from environmental samples. We provide systematic methods for environmental study design, enrichment, screening, and isolation of target microorganisms. Species identification is performed using qPCR or MALDI-TOF MS. Genomic DNA is extracted for whole-genome sequencing using the Oxford Nanopore platform. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Designing and conducting an environmental soil sampling study Basic Protocol 2: Enrichment of microbes from environmental soil samples Alternate Protocol 1: Collection and enrichment of microbes from environmental water samples Basic Protocol 3: Screening of enriched samples by direct qPCR Basic Protocol 4: Enumeration and isolation of enriched samples using selective medium Basic Protocol 5: Species confirmation using colony qPCR Alternate Protocol 2: Species identification using a MALDI-TOF MS Biotyper Alternate Protocol 3: Species identification of bacterial isolates using universal PCR primers and Sanger sequencing Basic Protocol 6: Cryostorage of bacterial isolates Basic Protocol 7: Extraction of genomic DNA Basic Protocol 8: Quality check of extracted genomic DNA Basic Protocol 9: Whole-genome sequencing using the Oxford Nanopore MinION Platform.
MeSH term(s)	DNA Primers ; Nanopores ; Polymerase Chain Reaction ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Soil
Chemical Substances	DNA Primers ; Soil
Language	English
Publishing date	2023-05-29
Publishing country	United States
Document type	Journal Article
ISSN	2691-1299
ISSN (online)	2691-1299
DOI	10.1002/cpz1.778
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: GALAXY Workflow for Bacterial Next-Generation Sequencing De Novo Assembly and Annotation.

Wee, Soon Keong / Yap, Eric Peng Huat

Current protocols

2021 Volume 1, Issue 9, Page(s) e242

Abstract: Whole-genome sequencing of prokaryotes is now readily available and affordable on next-generation sequencing platforms. However, the process of de novo assembly can be complicated and tedious for those without a background in computational biology, ... ...

Abstract	Whole-genome sequencing of prokaryotes is now readily available and affordable on next-generation sequencing platforms. However, the process of de novo assembly can be complicated and tedious for those without a background in computational biology, bioinformatics, or UNIX. Licenses for commercial bioinformatics software may be costly and limited in flexibility. GALAXY is a powerful graphical open-source code-free bioinformatics platform that is freely available on multiple public and private servers. Here, we describe a bacterial de novo assembly workflow using GALAXY. It performs de novo genome assembly using short reads, long reads, or a hybrid method using both short and long reads. Genome annotation, prediction of antimicrobial resistance genes, and multi-locus sequence typing are subsequently performed to characterize the draft genome. Performing genome assembly and annotation on this pipeline allows documentation, parameterization, and sharing, facilitating replication, reuse, and reproducibility of both data and methods. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Quality check of NGS reads Basic Protocol 2: De novo assembly using Unicycler Basic Protocol 3: Assembly quality check using QUAST and Bandage Basic Protocol 4: Genome annotation using Prokka Basic Protocol 5: Prediction of antimicrobial resistance genes (ARGs) Basic Protocol 6: Multi-locus sequence typing (MLST).
MeSH term(s)	High-Throughput Nucleotide Sequencing ; Multilocus Sequence Typing ; Reproducibility of Results ; Sequence Analysis, DNA ; Workflow
Language	English
Publishing date	2021-09-07
Publishing country	United States
Document type	Journal Article
ISSN	2691-1299
ISSN (online)	2691-1299
DOI	10.1002/cpz1.242
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Draft Genome Sequence of Enterobacter hormaechei subsp.

Wee, Soon Keong / Chan, Sharon Cui Mun / Guan, Xue Li / Yap, Eric Peng Huat

Microbiology resource announcements

2021 Volume 10, Issue 28, Page(s) e0040621

Abstract: Here, we report the genome sequence of Enterobacter hormaechei subsp. ...

Abstract	Here, we report the genome sequence of Enterobacter hormaechei subsp.
Language	English
Publishing date	2021-07-15
Publishing country	United States
Document type	Journal Article
ISSN	2576-098X
ISSN (online)	2576-098X
DOI	10.1128/MRA.00406-21
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler.

Wee, Soon Keong / Sivalingam, Suppiah Paramalingam / Yap, Eric Peng Huat

Genes

2020 Volume 11, Issue 6

Abstract: There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), ... ...

Abstract	There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.
MeSH term(s)	Betacoronavirus/genetics ; Betacoronavirus/isolation & purification ; COVID-19 ; Coronavirus Infections/diagnosis ; Coronavirus Infections/virology ; Exudates and Transudates/virology ; Humans ; Limit of Detection ; Nucleocapsid/genetics ; Pandemics ; Pneumonia, Viral/diagnosis ; Pneumonia, Viral/virology ; Point-of-Care Systems ; Polymerase Chain Reaction/methods ; RNA, Viral/analysis ; SARS-CoV-2 ; Sputum/virology
Chemical Substances	RNA, Viral
Keywords	covid19
Language	English
Publishing date	2020-06-18
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2527218-4
ISSN	2073-4425 ; 2073-4425
ISSN (online)	2073-4425
ISSN	2073-4425
DOI	10.3390/genes11060664
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler

Soon Keong Wee / Suppiah Paramalingam Sivalingam / Eric Peng Huat Yap

Genes, Vol 11, Iss 664, p

2020 Volume 664

Abstract	There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.
Keywords	point-of-care ; diagnostics ; DIRECT-PCR ; portable PCR ; SARS-CoV-2 ; Covid-19 ; Genetics ; QH426-470 ; covid19
Subject code	572
Language	English
Publishing date	2020-06-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

Wee, Soon Keong / Sivalingam, Suppiah Paramalingam / Yap, Eric Peng Huat

bioRxiv

Abstract	There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single-tube homogeneous reaction within 36 minutes. This minimized hands-on time, reduces turnaround-time for sample-to-result and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.
Keywords	covid19
Language	English
Publishing date	2020-04-20
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2020.04.17.042366
Database	COVID19

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Article: Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler

Wee, Soon Keong / Sivalingam, Suppiah Paramalingam / Yap, Eric Peng Huat

Genes. 2020 June 18, v. 11, no. 6

2020

Abstract	There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.
Keywords	COVID-19 infection ; RNA ; Severe acute respiratory syndrome coronavirus 2 ; cost effectiveness ; gene amplification ; genes ; nose ; nucleocapsid ; pandemic ; reverse transcriptase polymerase chain reaction ; reverse transcription ; screening
Language	English
Dates of publication	2020-0618
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2527218-4
ISSN	2073-4425
ISSN	2073-4425
DOI	10.3390/genes11060664
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

Wee, Soon Keong / Sivalingam, Suppiah Paramalingam / Yap, Eric Peng Huat

bioRxiv

Abstract	There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single-tube homogeneous reaction within 36 minutes. This minimized hands-on time, reduces turnaround-time for sample-to-result and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.
Keywords	covid19
Publisher	BioRxiv; WHO
Document type	Article ; Online
DOI	10.1101/2020.04.17.042366
Database	COVID19

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Article ; Online: Discordance of epidermal growth factor receptor mutation between primary lung tumor and paired distant metastases in non-small cell lung cancer: A systematic review and meta-analysis.

Lee, Chia Ching / Soon, Yu Yang / Tan, Char Loo / Koh, Wee Yao / Leong, Cheng Nang / Tey, Jeremy Chee Seong / Tham, Ivan Weng Keong

PloS one

2019 Volume 14, Issue 6, Page(s) e0218414

Abstract: Purpose: To evaluate the rate of discordance of epidermal growth factor receptor (EGFR) mutation between primary lung tumor and paired distant metastases in non-small-cell lung cancer (NSCLC).: Methods: We performed a meta-analysis of 17 studies (518 ...

Abstract	Purpose: To evaluate the rate of discordance of epidermal growth factor receptor (EGFR) mutation between primary lung tumor and paired distant metastases in non-small-cell lung cancer (NSCLC). Methods: We performed a meta-analysis of 17 studies (518 cases) assessing discordance rates of EGFR mutation in primary tumors and paired distant metastases. We performed subgroup analyses based on EGFR mutation status in primary tumor (mutant or wildtype), site of distant metastasis (bone, central nervous system (CNS) or lung/ pleural), methods of testing (direct sequencing or allele-specific testing) and timing of metastasis (synchronous or metachronous). Results: The overall discordance rate in EGFR mutation was low at 10.36% (95% CI = 4.23% to 18.79%) and varied widely between studies (I2 = 83.18%). The EGFR discordance rate was statistically significantly higher in bone metastases (45.49%, 95% CI = 14.13 to 79.02) than CNS (17.26%, 95% CI = 7.64 to 29.74; P = 0.002) and lung/ pleural metastases (8.17%, 95% CI = 3.35 to 14.85; P < 0.001). Subgroup analyses did not demonstrate any significant effect modification on the discordance rates by the EGFR mutation status in primary lung tumor, methods of testing and timing of metastasis. Conclusion: The overall discordance rate in EGFR mutation between primary lung tumor and paired distant metastases in NSCLC is low, although higher discordance rates were observed in bone metastases compared with CNS and lung/pleural metastases. Future studies assessing the impact of EGFR mutation discordance on treatment outcomes are required.
MeSH term(s)	Bone Neoplasms/genetics ; Bone Neoplasms/pathology ; Bone Neoplasms/secondary ; Carcinoma, Non-Small-Cell Lung/genetics ; Carcinoma, Non-Small-Cell Lung/pathology ; Carcinoma, Non-Small-Cell Lung/secondary ; Central Nervous System Neoplasms/genetics ; Central Nervous System Neoplasms/pathology ; Central Nervous System Neoplasms/secondary ; ErbB Receptors/genetics ; Humans ; Mutation ; Neoplasm Metastasis
Chemical Substances	EGFR protein, human (EC 2.7.10.1) ; ErbB Receptors (EC 2.7.10.1)
Language	English
Publishing date	2019-06-19
Publishing country	United States
Document type	Journal Article ; Meta-Analysis ; Systematic Review
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0218414
Database	MEDical Literature Analysis and Retrieval System OnLINE

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