Whole-Genome Sequencing Protocols for IBV and Other Coronaviruses Using High-Throughput Sequencing

2020 ◽  
pp. 67-74
Author(s):  
Graham L. Freimanis ◽  
Michael S. Oade
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Whole Genome

scholarly journals Whole Genome Sequencing of Enterovirus species C Isolates by High-Throughput Sequencing: Development of Generic Primers

2016 ◽  
Vol 7 ◽  
Author(s):  
Maël Bessaud ◽  
Serge A. Sadeuh-Mba ◽  
Marie-Line Joffret ◽  
Richter Razafindratsimandresy ◽  
Patsy Polston ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Whole Genome

scholarly journals Analysis of Aneuploidy Spectrum From Whole-Genome Sequencing Provides Rapid Assessment of Clonal Variation Within Established Cancer Cell Lines

2021 ◽  
Vol 20 ◽  
pp. 117693512110492
Author(s):  
Ahmed Ibrahim Samir Khalil ◽  
Anupam Chattopadhyay ◽  
Amartya Sanyal
Keyword(s):  
Whole Genome Sequencing ◽  
Cell Lines ◽  
Cancer Cell ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cancer Cell Lines ◽  
Easy Access ◽  
Clonal Variation ◽  
Whole Genome

Background: The revolution in next-generation sequencing (NGS) technology has allowed easy access and sharing of high-throughput sequencing datasets of cancer cell lines and their integrative analyses. However, long-term passaging and culture conditions introduce high levels of genomic and phenotypic diversity in established cell lines resulting in strain differences. Thus, clonal variation in cultured cell lines with respect to the reference standard is a major barrier in systems biology data analyses. Therefore, there is a pressing need for a fast and entry-level assessment of clonal variations within cell lines using their high-throughput sequencing data. Results: We developed a Python-based software, AStra, for de novo estimation of the genome-wide segmental aneuploidy to measure and visually interpret strain-level similarities or differences of cancer cell lines from whole-genome sequencing (WGS). We demonstrated that aneuploidy spectrum can capture the genetic variations in 27 strains of MCF7 breast cancer cell line collected from different laboratories. Performance evaluation of AStra using several cancer sequencing datasets revealed that cancer cell lines exhibit distinct aneuploidy spectra which reflect their previously-reported karyotypic observations. Similarly, AStra successfully identified large-scale DNA copy number variations (CNVs) artificially introduced in simulated WGS datasets. Conclusions: AStra provides an analytical and visualization platform for rapid and easy comparison between different strains or between cell lines based on their aneuploidy spectra solely using the raw BAM files representing mapped reads. We recommend AStra for rapid first-pass quality assessment of cancer cell lines before integrating scientific datasets that employ deep sequencing. AStra is an open-source software and is available at https://github.com/AISKhalil/AStra .

scholarly journals Whole Genome Sequencing of Enteroviruses Species A to D by High-Throughput Sequencing: Application for Viral Mixtures

2018 ◽  
Vol 9 ◽  
Author(s):  
Marie-Line Joffret ◽  
Patsy M. Polston ◽  
Richter Razafindratsimandresy ◽  
Maël Bessaud ◽  
Jean-Michel Heraud ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Whole Genome

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 Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides

Blood ◽  
2015 ◽  
Vol 126 (4) ◽  
pp. 508-519 ◽  
Author(s):  
Laura Y. McGirt ◽  
Peilin Jia ◽  
Devin A. Baerenwald ◽  
Robert J. Duszynski ◽  
Kimberly B. Dahlman ◽  
...  
Keyword(s):  
Cell Death ◽  
Whole Genome Sequencing ◽  
Effective Treatment ◽  
Mycosis Fungoides ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Whole Genome ◽  
Oncogenic Mutations ◽  
Key Points

Key Points High-throughput sequencing of MF revealed multiple mutations within epigenetic and cytokine pathways that may drive disease. Pharmacologically targeting the JAK3 pathway in MF results in cell death and may be an effective treatment of this disease.

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

Whole-exome and whole-genome sequencing in chronic lymphocytic leukemia: new biomarkers to target

2020 ◽  
Vol 21 (13) ◽  
pp. 957-962
Author(s):  
Charbel Hobeika ◽  
Gaelle Rached ◽  
Alain Chebly ◽  
Eliane Chouery ◽  
Hampig Raphael Kourie
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput Sequencing ◽  
Gene Mutations ◽  
Lymphocytic Leukemia ◽  
Whole Genome ◽  
Mutational Status ◽  
Whole Exome ◽  
Worse Prognosis

Many biomarkers indicate prognosis in chronic lymphocytic leukemia; such as fluorescence in situ hybridization testing: 17p or 11q deletions have a worse prognosis than trisomy 12, 13q deletion or normal result, or the mutational status of the immunoglobulin heavy chain (IGHV): unmutated IGHV have a worse prognosis than mutated IGHV. Recently, many gene mutations ( TP53, NOTCH1 etc.,) have been linked to a worse prognosis. With the new era of high-throughput sequencing, it has become easier to study gene mutations and their implication in predicting prognosis. In this review, we aim to review all the studies that performed whole-exome sequencing or whole-genome sequencing on chronic lymphocytic leukemia cells and explore the implication of various genes in disease prognosis.

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
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