scholarly journals Real-Time SARS-CoV-2 Genotyping by High-Throughput Multiplex PCR Reveals the Epidemiology of the Variants of Concern in Qatar

2021 ◽  
Author(s):  
Mohammad R. Hasan ◽  
Mahesh K. R. Kalikiri ◽  
Faheem Mirza ◽  
Sathyavathi Sundararaju ◽  
Anju Sharma ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Genome Sequencing ◽  
High Throughput ◽  
Sanger Sequencing ◽  
Transmission Dynamics ◽  
Whole Genome ◽  
Real Time Tracking ◽  
Alpha Beta ◽  
Pcr Genotyping

Complementing whole genome sequencing strategies with high-throughput multiplex RT-qPCR genotyping allows for more comprehensive and real-time tracking of SARS-CoV-2 variants of concern. During the second and third waves of COVID-19 in Qatar, PCR genotyping, combined with Sanger sequencing of un-typeable samples, was employed to describe the epidemiology of the Alpha, Beta and Delta variants. A total of 9792 nasopharyngeal PCR-positive samples collected between April-June 2021 were successfully genotyped, revealing the importation and transmission dynamics of these three variants in Qatar.

scholarly journals Discriminatory Weight of SNPs in Spike SARS-CoV-2 Variants: A Technically Rapid, Unambiguous, and Bioinformatically Validated Laboratory Approach

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 123
Author(s):  
Nicolò Musso ◽  
Paolo Giuseppe Bonacci ◽  
Dafne Bongiorno ◽  
Stefano Stracquadanio ◽  
Dalida Angela Bivona ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Sanger Sequencing ◽  
N Gene ◽  
Whole Genome ◽  
S Gene ◽  
Rapid Spread ◽  
Alpha Beta ◽  
Alpha Variant ◽  
Commercial Kits ◽  
Gene Portion

Background: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Methods: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). Results: A “weight” could be assigned to each mutation that may be present in the selected portion of the S gene. The method’s robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.

scholarly journals Molecular Detection of Carbapenemases in Enterobacterales: A Comparison of Real-Time Multiplex PCR and Whole-Genome Sequencing

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 726
Author(s):  
Katja Probst ◽  
Dennis Nurjadi ◽  
Klaus Heeg ◽  
Anne-Marie Frede ◽  
Alexander H. Dalpke ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Multiplex Pcr ◽  
Real Time ◽  
Genome Sequencing ◽  
Draft Genome ◽  
University Hospital ◽  
Detection Methods ◽  
Whole Genome ◽  
Carbapenem Resistant ◽  
Antimicrobial Resistance Gene

Carbapenem-resistant Enterobacterales are a growing problem in healthcare systems worldwide. While whole-genome sequencing (WGS) has become a powerful tool for analyzing transmission and possible outbreaks, it remains laborious, and the limitations in diagnostic workflows are not well studied. The aim of this study was to compare the performance of WGS and real-time multiplex PCR (RT-qPCR) for diagnosing carbapenem-resistant Enterobacterales. In this study, we analyzed 92 phenotypically carbapenem-resistant Enterobacterales, sent to the University Hospital Heidelberg in 2019, by the carbapenem inactivation method (CIM) and compared WGS and RT-qPCR as genotypic carbapenemase detection methods. In total, 80.4% of the collected isolates were identified as carbapenemase producers. For six isolates, discordant results were recorded for WGS, PCR and CIM, as the carbapenemase genes were initially not detected by WGS. A reanalysis using raw reads, rather than assembly, highlighted a coverage issue with failure to detect carbapenemases located in contigs with a coverage lower than 10×, which were then discarded. Our study shows that multiplex RT-qPCR and CIM can be a simple alternative to WGS for basic surveillance of carbapenemase-producing Enterobacterales. Using WGS in clinical workflow has some limitations, especially regarding coverage and sensitivity. We demonstrate that antimicrobial resistance gene detection should be performed on the raw reads or non-curated draft genome to increase sensitivity.

scholarly journals In-House, Rapid, and Low-Cost SARS-CoV-2 Spike Gene Sequencing Protocol to Identify Variants of Concern Using Sanger Sequencing

2021 ◽  
Author(s):  
Fatimah Alhamlan ◽  
Dana Bakheet ◽  
Marie Bohol ◽  
Madain Alsanea ◽  
Basma Alahaideb ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Rna Extraction ◽  
Whole Genome ◽  
Diagnostic Laboratory ◽  
Spike Gene

Background: The need for active genomic sequencing surveillance to rapidly identify circulating SARS-CoV-2 variants of concern (VOCs) is critical. However, increased global demand has led to a shortage of commercial SARS-CoV-2 sequencing kits, and not every country has the technological capability or the funds for high-throughput sequencing platforms. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol that uses supplies, equipment, and methodologic expertise available in standard molecular or diagnostic laboratories to identify circulating SARS-CoV-2 variants of concern. Methods: Sets of primers flanking the SARS-CoV-2 spike gene were designed using SARS-CoV-2 genome sequences retrieved from the Global Initiative on Sharing Avian Influenza Data (GISAID) Database and synthesized in-house. Primer specificity and final sequences were verified using online prediction analyses with BLAST. The primers were validated using 282 nasopharyngeal samples collected from patients assessed as positive for SARS-CoV-2 at the diagnostic laboratory of the hospital using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit. The patient samples were subjected to RNA extraction followed by cDNA synthesis, conventional polymerase chain reaction, and Sanger sequencing. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole genome sequencing of the same samples. Results: Sanger sequencing using the newly designed primers and next-generation whole genome sequencing of 282 patient samples indicated identical variants of concern results: 123 samples contained the alpha variant (B.1.1.7); 78, beta (B.1.351), 0, gamma (P.1), and 13, delta (B.1.617.2). Moreover, the remaining samples were non-VOC that belonged to none of these variants and had 99.97% identity with the reference genome. Only four samples had poor sequence quality by Sanger sequencing owing to a low viral count (Ct value >38). Therefore, mutation calls were >98% accurate. Conclusions: Sanger sequencing method using in-house primers is an alternative approach that can be used in facilities with existing equipment to mitigate limitations in high throughput supplies required to identify SARS-CoV-2 variants of concern during the COVID-19 pandemic. This protocol is easily adaptable for detection of emerging variants.

scholarly journals High-precision and cost-efficient sequencing for real-time COVID-19 surveillance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung Yong Park ◽  
Gina Faraci ◽  
Pamela M. Ward ◽  
Jane F. Emerson ◽  
Ha Youn Lee
Keyword(s):  
Los Angeles ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
High Precision ◽  
High Throughput Sequencing ◽  
Whole Genome ◽  
Sequencing Data ◽  
Public Health Response ◽  
Cost Efficient

AbstractCOVID-19 global cases have climbed to more than 33 million, with over a million total deaths, as of September, 2020. Real-time massive SARS-CoV-2 whole genome sequencing is key to tracking chains of transmission and estimating the origin of disease outbreaks. Yet no methods have simultaneously achieved high precision, simple workflow, and low cost. We developed a high-precision, cost-efficient SARS-CoV-2 whole genome sequencing platform for COVID-19 genomic surveillance, CorvGenSurv (Coronavirus Genomic Surveillance). CorvGenSurv directly amplified viral RNA from COVID-19 patients’ Nasopharyngeal/Oropharyngeal (NP/OP) swab specimens and sequenced the SARS-CoV-2 whole genome in three segments by long-read, high-throughput sequencing. Sequencing of the whole genome in three segments significantly reduced sequencing data waste, thereby preventing dropouts in genome coverage. We validated the precision of our pipeline by both control genomic RNA sequencing and Sanger sequencing. We produced near full-length whole genome sequences from individuals who were COVID-19 test positive during April to June 2020 in Los Angeles County, California, USA. These sequences were highly diverse in the G clade with nine novel amino acid mutations including NSP12-M755I and ORF8-V117F. With its readily adaptable design, CorvGenSurv grants wide access to genomic surveillance, permitting immediate public health response to sudden threats.

Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

2022 ◽  
Author(s):  
Jason Nguyen ◽  
Rebecca Hickman ◽  
Tracy Lee ◽  
Natalie Prystajecky ◽  
John Tyson
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Library Preparation ◽  
Dna Libraries ◽  
Reaction Volumes ◽  
Single Size ◽  
Stop Buffer

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed. This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e

scholarly journals High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae

2014 ◽  
Vol 14 (1) ◽  
pp. 49 ◽  
Author(s):  
Galina Sergeev ◽  
Sambit Roy ◽  
Michael Jarek ◽  
Viktor Zapolskii ◽  
Dieter E Kaufmann ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Vibrio Cholerae ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Screening ◽  
Whole Genome ◽  
Active Inhibitor

scholarly journals Genome Sequence ofListeria monocytogenesStrain HPB2088 (Serotype 1/2a), an Environmental Isolate Collected in Canada in 1994

2016 ◽  
Vol 4 (4) ◽  
Author(s):  
Arthur W. Pightling ◽  
Hugh Rand ◽  
Errol Strain ◽  
Franco Pagotto
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Genome Sequence ◽  
Foodborne Pathogen ◽  
Environmental Isolate ◽  
Severe Illness ◽  
Whole Genome ◽  
Serotype 1

Listeria monocytogenesis a foodborne pathogen that causes severe illness. Thus, ongoing efforts at real-time whole-genome sequencing are of utmost importance. However, it is also important that retrospective analyses that place these data into context be performed. Here, we present the genome sequence of strain HPB2088, which was collected in 1994.

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