SARS-CoV-2 whole-genome sequencing using reverse complement PCR: For easy, fast and accurate outbreak and variant analysis.

Abstract Whole-genome sequencing is increasingly being used to investigate the spatial and temporal distribution of viral pathogens including the Severe Acute Respiratory Syndrome Coronavirus Variant 2 (SARS-CoV-2) which is responsible for the ongoing COVID-19 pandemic. In this study, we determined 55 complete genome sequences of SARS-CoV-2 strains isolated from patients from Noakhali, a South-Eastern district in Bangladesh. Variant analysis of our sequenced genomes identified sixteen rare variations in S, six in N, two in M, one in E protein and the S protein variation, Y204F, identified in two of our sequenced strains, has not been reported from any other countries in the GISAID database. Comparison of the prevalence pattern across the country showed GH clade lineages B.1.36 and B.1.36.16 to be abundant in Noakhali and the South-Eastern region of Chittagong when compared to the rest of the country. Phylodynamic analysis of our sequenced genomes revealed that the virus was estimated to be evolving at the rate of 1.065 X 10− 4 subs/site/year. The study results demonstrated the necessity of initiating a concerted, country-wide genomics surveillance effort to determine any novel mutation of functional significance, understanding virus evolution, transmission, and spread in Bangladesh. Short running title: Genome sequencing of Noakhali isolates SARS-Cov-2 in Bangladesh

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Multiplex Fragment analysis detects all COVID-19 variants of concern

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab191.294 ◽

2021 ◽

Vol 156 (Supplement_1) ◽

pp. S138-S138

Author(s):

J A SoRelle ◽

A Clark ◽

Z Wang ◽

J Park

Keyword(s):

New York ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Fragment Analysis ◽

Deletion Mutations ◽

Percent Agreement ◽

Indian Origin ◽

Allele Specific ◽

Variant Analysis

Abstract Introduction/Objective The majority of tracking methods have employed whole genome sequencing, which can be very expensive and time consuming. An alternative method has been to use genotyping of specific mutations to identify variants. However, tracking SARS-CoV-2 variants by targeted methods has been a moving target. Most methods only multiplex four targets per reaction, but we have multiplexed 8 targets in a single tube using fragment analysis. Methods/Case Report Fluorescently labeled primers targeted a combination of insertion/ deletion mutations and single nucleotide mutations. The PCR amplified products, amplicons, were separated by capillary electrophoresis. Primers were designed to detect changes in size indicative of insertion or deletion mutations including: ORF1A:Del3675_3677, S:Del69_70, S:Del144, S:Del157_158, S:Del242_244, ORF8:Del119_120, and ORF8:ins28269-28273. Allele-specific primers were designed to detect both the wild-type and mutated versions of S:N501Y, S:E484K, and S:L452R. Residual nasopharyngeal and nasal specimens testing positive for SARS-CoV-2 by RT-PCR or isothermal amplification (IDnow) methods were selected from May 1- June 24, 2021. Variant analysis was performed by multiplex targeted PCR and whole genome sequencing in parallel on the same specimens to determine positive percent agreement. Results (if a Case Study enter NA) Variant analysis was performed on 250 specimens detecting each of the major variants of concern Alpha (B.1.1.7, U.K. origin, n= 108), Beta (B.1.351, South Africa origin, n=3), Gamma (P.1, Brazil origin, n=12), Delta (B.1.617.2, Indian origin, n=17), and Iota (B.1.526, New York, n=5). Some specimens with low viral load were detected by only PCR (n=18), only WGS (n=41), or neither (n=20). Overall positive percent agreement was 95% (163/171). Conclusion This adjustable method robustly and accurately identifies COVID-19 VOCs utilizing a platform amenable to multiple targets (20-40 targets ranging from 100-500b.p. across four fluorescent channels) using equipment commonly found in routine molecular pathology laboratories. Future directions include adjusting targets to detect new variants.

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Whole-Genome sequencing and genetic variant analysis of a quarter Horse mare

BMC Genomics ◽

10.1186/1471-2164-13-78 ◽

2012 ◽

Vol 13 (1) ◽

pp. 78 ◽

Cited By ~ 43

Author(s):

Ryan Doan ◽

Noah D Cohen ◽

Jason Sawyer ◽

Noushin Ghaffari ◽

Charlie D Johnson ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genetic Variant ◽

Whole Genome ◽

Quarter Horse ◽

Variant Analysis

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Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease

The American Journal of Human Genetics ◽

10.1016/j.ajhg.2016.12.003 ◽

2017 ◽

Vol 100 (1) ◽

pp. 75-90 ◽

Cited By ~ 169

Author(s):

Keren J. Carss ◽

Gavin Arno ◽

Marie Erwood ◽

Jonathan Stephens ◽

Alba Sanchis-Juan ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Rare Variant ◽

Molecular Pathology ◽

Retinal Disease ◽

Whole Genome ◽

Rare Variant Analysis ◽

Inherited Retinal Disease ◽

Variant Analysis

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Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables easy, fast and accurate outbreak analysis in hospital and community settings

10.1101/2020.10.29.360578 ◽

2020 ◽

Author(s):

Femke Wolters ◽

Jordy P.M. Coolen ◽

Alma Tostmann ◽

Lenneke F.J. van Groningen ◽

Chantal P. Bleeker-Rovers ◽

...

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Healthcare Workers ◽

Whole Genome ◽

Sequencing Technology ◽

Reverse Complement ◽

Oxford Nanopore ◽

Epidemiological Link ◽

Oxford Nanopore Technologies

AbstractBackgroundCurrent transmission rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still increasing and many countries are facing second waves of infections. Rapid SARS-CoV-2 whole-genome sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring and determining transmission links. Here we report the use of reverse complement polymerase chain reaction (RC-PCR), a novel technology for WGS of SARS-CoV-2 enabling library preparation in a single PCR saving time, resources and enables high throughput screening. Additionally, we show SARS-CoV-2 diversity and possible transmission within the Radboud university medical center (Radboudumc) during September 2020 using RC-PCR WGS.MethodsA total of 173 samples tested positive for SARS-CoV-2 between March and September 2020 were selected for whole-genome sequencing. Ct values of the samples ranged from 16 to 42. They were collected from 83 healthcare workers and three patients at the Radboudumc, in addition to 64 people living in the area around the hospital and tested by the local health services. For validation purposes, nineteen of the included samples were previously sequenced using Oxford Nanopore Technologies and compared to RC-PCR WGS results. The applicability of RC-PCR WGS in outbreak analysis for public health service and hospitals was tested on six suspected clusters containing samples of healthcare workers and patients with an epidemiological link.FindingsRC-PCR resulted in sequencing data for 146 samples. It showed a genome coverage of up to 98,2% for samples with a maximum Ct value of 32. Comparison to Oxford Nanopore technologies gives a near-perfect agreement on 95% of the samples (18 out of 19). Three out of six clusters with a suspected epidemiological link were fully confirmed, in the others, four healthcare workers were not associated. In the public health service samples, a previously unknown chain of transmission was confirmed.Significance statementSAR-CoV-2 whole-genome sequencing using RC-PCR is a reliable technique and applicable for use in outbreak analysis and surveillance. Its ease of use, high-trough screening capacity and wide applicability makes it a valuable addition or replacement during this ongoing SARS-CoV-2 pandemic.FundingNoneResearch in contextEvidence before this studyAt present whole genome sequencing techniques for SARS-CoV-2 have a large turnover time and are not widely available. Only a few laboratories are currently able to perform large scale SARS-CoV-2 sequencing. This restricts the use of sequencing to aid hospital and community infection prevention.Added value of this studyHere we present clinical and technical data on a novel Whole Genome Sequencing technology, implementing reverse-complement PCR. It is able to obtain high genome coverage of SARS-CoV-2 and confirm and exclude epidemiological links in 173 healthcare workers and patients. The RC-PCR technology simplifies the workflow thereby reducing hands on time. It combines targeted PCR and sequence library construction in a single PCR, which normally takes several steps. Additionally, this technology can be used in concordance with the widely available range of Illumina sequencers.Implications of all the available evidenceRC-PCR whole genome sequencing technology enables rapid and targeted surveillance and response to an ongoing outbreak that has great impact on public health and society. Increased use of sequencing technologies in local laboratories can help prevent increase of SARS-CoV-2 spreading by better understanding modes of transmission.

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Whole genome sequencing, variant analysis, phylogenetics, and deep sequencing of Zika virus strains

Scientific Reports ◽

10.1038/s41598-018-34147-7 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 9

Author(s):

Susmita Shrivastava ◽

Vinita Puri ◽

Kari A. Dilley ◽

Erica Ngouajio ◽

Jessica Shifflett ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Deep Sequencing ◽

Zika Virus ◽

Whole Genome ◽

Variant Analysis ◽

Virus Strains

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STAAR Workflow: A cloud-based workflow for scalable and reproducible rare variant analysis

10.1101/2021.09.07.456116 ◽

2021 ◽

Author(s):

Sheila M. Gaynor ◽

Kenneth E. Westerman ◽

Lea L. Ackovic ◽

Xihao Li ◽

Zilin Li ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Rare Variant ◽

Rare Variants ◽

Association Studies ◽

Whole Genome ◽

Sequencing Analysis ◽

Functional Annotations ◽

Rare Variant Analysis ◽

Variant Analysis

AbstractSummaryWe developed the STAAR WDL workflow to facilitate the analysis of rare variants in whole genome sequencing association studies. The open-access STAAR workflow written in the workflow description language (WDL) allows a user to perform rare variant testing for both gene-centric and genetic region approaches, enabling genome-wide, candidate, and conditional analyses. It incorporates functional annotations into the workflow as introduced in the STAAR method in order to boost the rare variant analysis power. This tool was specifically developed and optimized to be implemented on cloud-based platforms such as BioData Catalyst Powered by Terra. It provides easy-to-use functionality for rare variant analysis that can be incorporated into an exhaustive whole genome sequencing analysis pipeline.Availability and implementationThe workflow is freely available from https://dockstore.org/workflows/github.com/sheilagaynor/STAAR_workflow.

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