Rapid High Throughput Whole Genome Sequencing of SARS-CoV-2 by using One-step RT-PCR Amplification with Integrated Microfluidic System and Next-Gen Sequencing

The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here we report a simple and efficient workflow for whole genome sequencing utilizing one-step RT-PCR amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm Integrated Fluidic Circuit (IFC) and instruments to amplify 48 samples with 39 pairs of primers, including 35 custom designed primer pairs and four additional primer pairs from the ARTIC network protocol v3. Application of this method on RNA samples from both viral isolate and clinical specimens demonstrate robustness and efficiency of this method in obtaining the full genome sequence of SARS-CoV-2.

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Rapid High Throughput Whole Genome Sequencing of SARS-CoV-2 by using One-step RT-PCR Amplification with Integrated Microfluidic System and Next-Gen Sequencing

10.1101/2020.11.04.369165 ◽

2020 ◽

Author(s):

Tao Li ◽

Hye Kyung Chung ◽

Papa K. Pireku ◽

Brett F. Beitzel ◽

Mark A. Sanborn ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Pcr Amplification ◽

Amplicon Sequencing ◽

Microfluidic System ◽

Single Step ◽

Full Genome Sequence ◽

Whole Genome ◽

Rt Pcr ◽

One Step

ABSTRACTThe long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here we report a simple and efficient workflow for whole genome sequencing utilizing one-step RT-PCR amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm IFC and instruments to amplify 48 samples with 39 pairs of primers in a single step. Application of this method on RNA samples from both viral isolate and clinical specimens demonstrate robustness and efficiency of this method in obtaining the full genome sequence of SARS-CoV-2.

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Molecular Investigation of an Ontario Mumps Outbreak using Whole Genome Sequencing

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx163.870 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S359-S359

Author(s):

Patrick Stapleton ◽

Alireza Eshaghi ◽

Eddie Chong-King ◽

Mark Cardona ◽

Steve Masney ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Fragment Size ◽

Pcr Amplification ◽

Mumps Virus ◽

Likelihood Method ◽

Cdna Libraries ◽

Whole Genome ◽

Rt Pcr ◽

Distinct Genotype

Abstract Background In early 2017 an outbreak of Mumps virus affected over 100 individuals in the province of Ontario, concurrent with multiple mumps virus outbreaks across North America. Traditional genotyping of mumps outbreaks relies on sequencing a portion of the small hydrophobic (SH) gene, but has limited capability to distinguish between strains of the same genotype. Most mumps cases in Ontario in recent years are of genotype G. We used a novel whole genome sequencing (WGS) protocol to perform a molecular epidemiological investigation of the outbreak. Methods Throat (n = 5) and buccal (n = 15) swabs positive by RT-PCR for SH or Fusion (F) gene targets were cultured in primary Rhesus monkey kidney cells. Cell free viral extract underwent RT-PCR and subsequent PCR amplification using overlapping primer pairs to cover the entire 15 kilobase (kb) genome. The first 8 samples were amplified with 18 pairs of overlapping primers, which was reduced to 9 sets (average fragment size 1.9 kb, range 1.6–2.8 kb) for the final 12 samples. Mumps cDNA libraries were prepared with Nextera XT kit and WGS of the indexed fragments was performed with V2 reagent kits on the Illumina MiSeq instrument. Reference based genome assembly was performed using samtools version 1.4. Phylogenetic analysis was performed by maximum likelihood method in MEGA7. Results We identified two distinct genotype G lineages comprised of 9 patients each and closely related to a 2009–2010 outbreak in Ontario and New York (Figure 1). Inter-lineage single nucleotide polymorphism (SNP) differences ranged from 25 to 31, whereas intra-lineage SNPs ranged from 0 to 8 SNPs. Two outlying sequences, of genotype C and G respectively, may represent sporadic introduction of virus from other areas. Time from virus isolation to SNP based analysis was approximately 4 days. Conclusion WGS of Mumps virus culture isolates using the PCR fragment method identified two distinct genotype G lineages in a large provincial outbreak. This method may aid public health authorities identify separate transmission chains in the case of large outbreaks. Disclosures All authors: No reported disclosures.

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False COVID-19 cases due to contamination by inactivated virus vaccine

Clinical Infectious Diseases ◽

10.1093/cid/ciab684 ◽

2021 ◽

Author(s):

Kelvin Kai-Wang To ◽

Xin Li ◽

David Christopher Lung ◽

Jonathan Daniel Ip ◽

Wan-Mui Chan ◽

...

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Virus Vaccine ◽

False Positive ◽

Spike Protein ◽

Whole Genome ◽

Rt Pcr ◽

Health Measures ◽

Respiratory Specimens

Abstract A false-positive SARS-CoV-2 RT-PCR result can lead to unnecessary public-health measures. We report two individuals whose respiratory specimens were contaminated by inactivated SARS-CoV-2 vaccine strain(CoronaVac), likely at vaccination premises. Incidentally, whole-genome sequencing of CoronaVac showed adaptive deletions on the spike protein, which do not result in observable changes of antigenicity.

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Next generation whole genome sequencing of Plasmodium falciparum using NextSeq500 technology in India

10.1101/068676 ◽

2016 ◽

Author(s):

Alassane Mbengue ◽

Pragya Namdev ◽

Tarkeshwar Kumar ◽

Kasturi Haldar ◽

Souvik Bhattacharjee

Keyword(s):

Plasmodium Falciparum ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Immune Evasion ◽

Malaria Elimination ◽

Protozoan Parasite ◽

Amplicon Sequencing ◽

Whole Genome ◽

Next Generation ◽

New Genes

AbstractPlasmodium falciparum is a protozoan parasite that causes the deadliest form of human malaria. Although, malaria burdens worldwide have decreased substantially over the last decade (WHO, 2014), genetic variation and adaptation by parasite strains against drugs and vaccines present significant challenges for the elimination of malaria (Ariey et al., 2014; Neafsey et al., 2015). India has formally launched a malaria elimination campaign (NVBDCP, 2016). Therefore, early in-country detection of drug resistance and/or immune evasion will be important for the program. Presently, the majority of surveillance methods in India detect a limited number of known polymorphisms (Campino et al., 2011; Chatterjee et al., 2016; Daniels et al., 2008; Mishra et al., 2015; Neafsey et al., 2012; Neafsey et al., 2008). A recently reported amplicon sequencing method enables targeted re-sequencing of a panel of genes (Rao et al., 2016). However, the capacity to identify new genes of resistance/immune evasion by whole genome sequencing (WGS) through next generation sequencing (NGS) in India, has remained elusive. Here we report the first WGS of P. falciparum strain performed by Eurofins Genomics India Pvt. Ltd at its Bengaluru division within 40 days of sample submission. Our data establish that timely, commercial WGS through NGS in India can be applied to P. falciparum to greatly empower the malaria elimination agenda in India.

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Detection of SARS-CoV-2 from Patient Fecal Samples by Whole Genome Sequencing

10.21203/rs.3.rs-48863/v1 ◽

2020 ◽

Author(s):

Andreas Papoutsis ◽

Thomas Borody ◽

Siba Dolai ◽

Jordan Daniels ◽

Skylar Steinberg ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Analysis ◽

Nasopharyngeal Swab ◽

Whole Genome ◽

Rt Pcr ◽

Whole Genome Analysis ◽

Fecal Samples ◽

Oral Transmission ◽

Study Participants

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment NGS from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal-oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication.

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SARS-CoV-2 N501Y introductions and transmissions in Switzerland from beginning of October 2020 to February 2021 – implementation of Swiss-wide diagnostic screening and whole genome sequencing

10.1101/2021.02.11.21251589 ◽

2021 ◽

Cited By ~ 2

Author(s):

Ana Rita Goncalves Cabecinhas ◽

Tim Roloff ◽

Madlen Stange ◽

Claire Bertelli ◽

Michael Huber ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Implementation Process ◽

Amplicon Sequencing ◽

World Health ◽

Whole Genome ◽

Diagnostic Systems ◽

Early Introduction ◽

Specific Pcr ◽

Health Organization

AbstractThe rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations by the European Center for Disease Prevention and Control (ECDC) and World Health Organization (WHO) for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological definitions based on travel history and the S gene dropout in certain diagnostic systems. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 3492 VoCs have been identified since the detection of the first Swiss case in October 2020, with 1370 being B1.1.7, 61 B.1.351, and none P.1. The remaining 2061 cases of VoCs have been described without further lineage specification. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs.

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Identification of nsp1 gene as the target of SARS‐CoV‐2 real‐time RT‐PCR using nanopore whole‐genome sequencing

Journal of Medical Virology ◽

10.1002/jmv.26140 ◽

2020 ◽

Vol 92 (11) ◽

pp. 2725-2734 ◽

Cited By ~ 4

Author(s):

Wan‐Mui Chan ◽

Jonathan Daniel Ip ◽

Allen Wing‐Ho Chu ◽

Cyril Chik‐Yan Yip ◽

Lap‐Sum Lo ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Real Time ◽

Genome Sequencing ◽

Whole Genome ◽

Rt Pcr ◽

Nsp1 Gene

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Assessment of Ion S5 NGS protocol for SARS-CoV-2 genome sequencing

Genetics & Applications ◽

10.31383/ga.vol5iss2pp24-30 ◽

2021 ◽

Vol 5 (2) ◽

pp. 24

Author(s):

Dino Pećar ◽

Ivana Čeko ◽

Lana Salihefendić ◽

Rijad Konjhodžić

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Magnetic Beads ◽

Rna Isolation ◽

Whole Genome ◽

Library Preparation ◽

Rt Pcr ◽

Sequence Coverage ◽

Cdna Synthesis ◽

Good Laboratory

Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.

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Emergence of SARS-CoV-2 variant B.1.575.2 containing the E484K mutation in the spike protein in Pamplona (Spain) May-June 2021

10.1101/2021.08.11.21261950 ◽

2021 ◽

Author(s):

Camino Trobajo-Sanmartín ◽

Ana Miqueleiz ◽

María Eugenia Portillo ◽

Miguel Fernández-Huerta ◽

Ana Navascués ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Threshold Value ◽

Spike Protein ◽

Whole Genome ◽

Viral Dynamics ◽

Rt Pcr ◽

Northern Spain ◽

Novel Mutations ◽

Rapid Control

With the emergence of new SARS-CoV-2 variants and the acquisition of novel mutations in exiting lineages, the need to implement methods capable of monitoring viral dynamics arises. We report the emergence and spread of a new SARS-CoV-2 variant within B.1.575 lineage containing the E484K mutation in the spike protein (named B.1.575.2) in a region of Northern Spain between May and June 2021. SARS-CoV-2 positive samples with cycle threshold value less than or equal to 30 were selected to screen of presumptive variants using the TaqPathTM COVID-19 RT-PCR kit and TaqManTM SARS-CoV-2 Mutation Panel. Confirmation of variants was performed by whole genome sequencing. Of the 200 samples belonging to the B.1.575 lineage, 194 (97%) corresponded to the B.1.575.2 sub-lineage, which was related to the presence of the E484K mutation. Of 197 cases registered in GISAID EpiCoV database as lineage B.1.575.2 194 (99.5%) were identified in Pamplona (Spain). This report emphasizes the importance of complementing surveillance of SARS-CoV-2 with sequencing for the rapid control of emerging viral variants.

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Optimization of multiplex PCR composition to screen for SARS-CoV-2 variants of concern

Acta Biochimica Indonesiana ◽

10.32889/actabioina.58 ◽

2021 ◽

Vol 4 (2) ◽

pp. 58

Author(s):

Maya Savira ◽

Enikarmila Asni ◽

Rahmat Azhari Kemal

Keyword(s):

Whole Genome Sequencing ◽

Multiplex Pcr ◽

Genome Sequencing ◽

Screening Method ◽

Positive Control ◽

Whole Genome ◽

Rt Pcr ◽

Pcr Screening ◽

Single Target ◽

Alpha Variant

Background: The ongoing COVID-19 pandemic has led to the emergence of several variants of concern. To rapidly identify those variants, screening samples for whole-genome sequencing (WGS) prioritization could be performed. Objective: We optimized the polymerase chain reaction (PCR) screening method to identify the mutation in spike and ORF1a regions. Methods: We adopted primers targeting mutation in spike and ORF1a region from another study. We optimized the PCR screening method using kits readily available in Indonesia. Firstly, we compared N1 and N2 primers as internal positive control. We also compared GoTaq® 1-Step RT-qPCR System and Indonesia TFRIC-19 BioCOV-19 for the multiplex reaction. We used the optimized composition to screen SARS-CoV-2 positive samples from April – June 2021. Samples with spike and/or ORF1a target failure were subjected to whole genome sequencing (WGS). Results: The results demonstrated the N2 BioCOV-19 reaction as the optimized multiplex PCR composition for spike and ORF1a mutations screening. Whole-genome sequencing has shown that a sample with spike and ORF1a targets failure to be Alpha variant, while other samples with single target failure as non-variants of concern. Therefore, a multiplex RT-PCR composition has been optimized to detect mutation in spike and ORF1a regions. Conclusion: We have optimized a multiplex RT-PCR composition to detect mutation in spike and ORF1a regions.

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