Pathogen-Oriented Low-cost Assembly & Re-sequencing (POLAR): A highly sensitive and high-throughput SARS-CoV-2 diagnostic based on whole genome sequencing v1 (protocols.io.bearjad6)

protocols.io ◽  
2020 ◽  
Author(s):  
Brian Glenn ◽  
Neva C ◽  
Namita Mitra ◽  
Saul Godinez ◽  
Ragini Mahajan ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Low Cost ◽  
Whole Genome ◽  
Highly Sensitive

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.

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 Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples

2016 ◽  
Author(s):  
Antonina A. Votintseva ◽  
Phelim Bradley ◽  
Louise Pankhurst ◽  
Carlos del Ojo Elias ◽  
Matthew Loose ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antibiotic Susceptibility ◽  
Point Of Care ◽  
Low Cost ◽  
Illumina Miseq ◽  
Turnaround Time ◽  
Reference Laboratory ◽  
Whole Genome ◽  
Specific Pcr

AbstractRoutine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.We demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a cost of £96/£198 per sample.We then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture-negative sputum DNA and BCG DNA. For the latest flowcell, the estimated turnaround time from patient to identification of BCG was 6 hours, with full susceptibility and surveillance results 2 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.

scholarly journals A High-Throughput Skim-sequencing Approach for Genotyping, Dosage Estimation and Identifying Translocations

2021 ◽  
Author(s):  
Laxman Adhikari ◽  
Sandesh Shrestha ◽  
Shuanyge Wu ◽  
Jared Crain ◽  
Liangliang Gao ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
High Throughput ◽  
Low Cost ◽  
Introgression Lines ◽  
Whole Genome ◽  
Sequencing Data ◽  
Genomic Libraries ◽  
Genetics Research ◽  
Large Populations ◽  
Sequencing Platforms

Abstract The development of next generation sequencing (NGS) enabled a shift from array-based genotyping to high-throughput genotyping by directly sequencing genomic libraries. Even though whole genome sequencing was initially too costly for routine analysis in large populations, such as those utilized for breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to utilize whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage, a limitation comes in the time and high cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on amount of data required and extended to 3,072 samples or more. Panels of double haploid wheat lines (Triticum aestivum, CDC Stanley x CDC Landmark), wheat-barley (T. aestivum x Hordeum vulgare) and wheat-wheatgrass (Triticum durum x Thinopyrum intermedium) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1x down to 0.01x per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the low coverage skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

scholarly journals Multiplex SARS-CoV-2 Genotyping PCR for Population-Level Variant Screening and Epidemiologic Surveillance

2021 ◽  
Author(s):  
Hannah Wang ◽  
Jacob A. Miller ◽  
Michelle Verghese ◽  
Mamdouh Sibai ◽  
Daniel Solis ◽  
...  
Keyword(s):  
Public Health ◽  
Nucleic Acid ◽  
Whole Genome Sequencing ◽  
San Francisco ◽  
Genome Sequencing ◽  
High Throughput ◽  
Population Level ◽  
Nucleic Acid Amplification ◽  
Whole Genome ◽  
Epidemiologic Surveillance

ABSTRACTBackgroundEmergence of SARS-CoV-2 variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a spike genotyping nucleic acid amplification test (NAAT) could facilitate high-throughput variant surveillance.MethodsWe designed and analytically validated a one-step multiplex allele-specific reverse transcriptase polymerase chain reaction (RT-qPCR) to detect three non-synonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2 positive specimens from our San Francisco Bay Area population.ResultsBetween December 1, 2020 and March 1, 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K+N501Y mutations. The assay had near-perfect (98-100%) concordance with whole-genome sequencing in a validation subset of 229 specimens, and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed rapid emergence of L452R in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021.ConclusionsWe developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.Summary / Key PointsEmergence of SARS-CoV-2 variants with concerning phenotypes is of public health interest. We developed a multiplex genotyping RT-qPCR to rapidly detect L452R, E484K, and N501Y with high sequencing concordance. This high-throughput alternative to resource-intensive sequencing enabled surveillance of L452R emergence.

scholarly journals Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples

2017 ◽  
Vol 55 (5) ◽  
pp. 1285-1298 ◽  
Author(s):  
Antonina A. Votintseva ◽  
Phelim Bradley ◽  
Louise Pankhurst ◽  
Carlos del Ojo Elias ◽  
Matthew Loose ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antibiotic Susceptibility ◽  
Point Of Care ◽  
Low Cost ◽  
Illumina Miseq ◽  
Reference Laboratory ◽  
Whole Genome ◽  
Content Type ◽  
Phylogenetic Placement

ABSTRACT Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

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