scholarly journals A Novel High-Throughput Nanopore-Sequencing-Based Strategy for Rapid and Automated S-Protein Typing of SARS-CoV-2 Variants

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2548
Author(s):  
Gabriel E. Wagner ◽  
Massimo G. Totaro ◽  
André Volland ◽  
Michaela Lipp ◽  
Sabine Saiger ◽  
...  
Keyword(s):  
High Throughput ◽  
Immune Escape ◽  
Low Cost ◽  
Amino Acid Level ◽  
Region Of Interest ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Protein Variant ◽  
S Protein ◽  
S Gene

Rapid molecular surveillance of SARS-CoV-2 S-protein variants leading to immune escape and/or increased infectivity is of utmost importance. Among global bottlenecks for variant monitoring in diagnostic settings are sequencing and bioinformatics capacities. In this study, we aimed to establish a rapid and user-friendly protocol for high-throughput S-gene sequencing and subsequent automated identification of variants. We designed two new primer pairs to amplify only the immunodominant part of the S-gene for nanopore sequencing. Furthermore, we developed an automated “S-Protein-Typer” tool that analyzes and reports S-protein mutations on the amino acid level including a variant of concern indicator. Validation of our primer panel using SARS-CoV-2-positive respiratory specimens covering a broad Ct range showed successful amplification for 29/30 samples. Restriction to the region of interest freed sequencing capacity by a factor of 12–13, compared with whole-genome sequencing. Using either the MinION or Flongle flow cell, our sequencing strategy reduced the time required to identify SARS-CoV-2 variants accordingly. The S-Protein-Typer tool identified all mutations correctly when challenged with our sequenced samples and 50 deposited sequences covering all VOCs (December 2021). Our proposed S-protein variant screening offers a simple, more rapid, and low-cost entry into NGS-based SARS-CoV-2 analysis, compared with current whole-genome approaches.

scholarly journals Exploring Quantitative Metagenomics Studies using Oxford Nanopore Sequencing: A Computational and Experimental Protocol

2020 ◽  
Author(s):  
Rohia ALILI ◽  
Eugeni BELDA ◽  
Karine CLEMENT ◽  
Phuong Le ◽  
Edi PRIFTI ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Gut Microbiome ◽  
Large Scale ◽  
Low Cost ◽  
Read Length ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Experimental Protocol ◽  
Microbial Composition ◽  
Oxford Nanopore

Abstract Background: The gut microbiome plays a major role in chronic diseases, several of which are characterized by an altered diversity and composition of bacterial communities. Large-scale sequencing projects allowed the characterization of these microbial community perturbations. However, a gap remains in how these discoveries can be translated into clinical applications. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed.Results: Here, we propose a computational and experimental protocol for whole genome quantitative metagenomics studies of the human gut microbiome with Oxford Nanopore sequencing technology (ONT). We developed a bioinformatic pipeline to process ONT sequences based on the evaluation of different alignment parameters in the estimation of microbial diversity and composition. We also optimized stool collection and DNA extraction methods to maximize read length, a critical parameter for the sequence alignment and classification. Our analytical pipeline was evaluated using simulations of metagenomic communities to reflect naturally occuring compositional variations. We then validated our experimental and analytical pipeline with stool samples from a bariatric surgery cohort sequenced with ONT and Illumina, revealing comparable diversity and microbial composition profiles. These results were compared to those previously obtained with SOLiD sequencing, where differences were observed, possibly explained by variations in library preparation steps. Finally, we found that sequences obtained with ONT allowed assembly of complete genomes for disease-related species.Conclusion: This protocol can be implemented in the clinical or individual setting, bringing rapid personalized whole genome profiling of target microbiome species. Keywords: quantitative metagenomics, microbiome, obesity, gut microbiota, microbial DNA extraction, sequencing, Simulation, Oxford Nanopore Technologies, MinION.

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 Symptomatic reinfection of SARS-CoV-2 with spike protein variant N440K associated with immune escape

2021 ◽  
Author(s):  
Pallavali Roja Rani ◽  
Mohamed Imran ◽  
Juturu Vijaya Lakshmi ◽  
Bani Jolly ◽  
Abhinav Jain ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Healthcare Worker ◽  
Genome Sequencing ◽  
South India ◽  
Immune Escape ◽  
Spike Protein ◽  
North India ◽  
Whole Genome ◽  
Protein Variant ◽  
Escape Variant

Here we describe a case of re-infection in an individual from South India characterized by whole genome sequencing of the virus isolated from both episodes. The analysis shows the presence of an immune escape variant N440K in the Spike protein in both episodes of infection. Incidentally, this variant was also found in a case of reinfection previously reported by us in a healthcare worker from North India

scholarly journals S Gene Target Failure (SGTF) in Commercial Multiplex RT-PCR assay as indicator to detect SARS-CoV-2 VOC B.1.1.7 lineage in Tamil Nadu, India

2021 ◽  
Author(s):  
Vidhya N M ◽  
Kumaresan A ◽  
Kalaivani V ◽  
Rajesh Kumar A ◽  
Gurunathan Subramanian ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Tamil Nadu ◽  
Immune Escape ◽  
Whole Genome ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Gene Target ◽  
S Gene ◽  
Significant Public Health

Emergence of Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) Variants of Concern (VOC) possessing improved virulence, transmissibility and/or immune-escape capabilities has raised significant public health concerns. In order to identify VOCs, WHO recommends Whole-Genome Sequencing approach, which is costly and involves longer completion time. Hence, potential role of commercial multiplex RT-PCR kit to screen variants rapidly is being attempted in this study. A total of 1200 suspected COVID samples from different districts of Tamil Nadu State (India) were screened with Thermo TaqPath RT-PCR kit and Altona Realstar RT-PCR Assay kit. Among 1200 screened, S-gene target failure (SGTF) phenomenon were identified in 112 samples while testing with TaqPath RT-PCR Kit. 100% concordant results were observed between SGTF phenomenon and whole-genome sequencing (WGS) results in detecting SARS-CoV-2 VOC B.1.1.7. TaqPath RT-PCR assay testing can be utilized by laboratories to screen rapidly the VOC B.1.1.7 variants, thus enabling early detection of B.1.1.7 variant infection and transmission in population. This in turn will pave way to implement suitable preventive measures by appropriate authorities to control the transmission of the viral variant.

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 Exploring Semi-Quantitative Metagenomic Studies Using Oxford Nanopore Sequencing: A Computational and Experimental Protocol

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1496
Author(s):  
Rohia Alili ◽  
Eugeni Belda ◽  
Phuong Le ◽  
Thierry Wirth ◽  
Jean-Daniel Zucker ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Large Scale ◽  
Low Cost ◽  
Extraction Methods ◽  
Read Length ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Experimental Protocol ◽  
Microbial Composition ◽  
Oxford Nanopore

The gut microbiome plays a major role in chronic diseases, of which several are characterized by an altered composition and diversity of bacterial communities. Large-scale sequencing projects allowed for characterizing the perturbations of these communities. However, translating these discoveries into clinical applications remains a challenge. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed. Here, we propose a computational and experimental protocol for whole-genome semi-quantitative metagenomic studies of human gut microbiome with Oxford Nanopore sequencing technology (ONT) that could be applied to other microbial ecosystems. We developed a bioinformatics protocol to analyze ONT sequences taxonomically and functionally and optimized preanalytic protocols, including stool collection and DNA extraction methods to maximize read length. This is a critical parameter for the sequence alignment and classification. Our protocol was evaluated using simulations of metagenomic communities, which reflect naturally occurring compositional variations. Next, we validated both protocols using stool samples from a bariatric surgery cohort, sequenced with ONT, Illumina, and SOLiD technologies. Results revealed similar diversity and microbial composition profiles. This protocol can be implemented in a clinical or research setting, bringing rapid personalized whole-genome profiling of target microbiome species.

scholarly journals Exploring Quantitative Metagenomics Studies Using Oxford Nanopore Sequencing: A Computational and Experimental Protocol

2021 ◽  
Author(s):  
Rohia Alili ◽  
Eugeni Belda ◽  
Phuong Le ◽  
Thierry Wirth ◽  
Jean-Daniel Zucker ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Large Scale ◽  
Low Cost ◽  
Extraction Methods ◽  
Read Length ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Experimental Protocol ◽  
Microbial Composition ◽  
Oxford Nanopore

Background: The gut microbiome plays a major role in chronic diseases, of which several are characterized by an altered composition and diversity of bacterial communities. Large-scale sequencing projects allowed characterizing the perturbations of these communities. However, translating these discoveries into clinical applications remains a challenges. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed. Results: Here, we propose a computational and experimental protocol for whole genome quantitative metagenomics studies of human gut microbiome with Oxford Nanopore sequencing technology (ONT) that could be applied to other microbial ecosystems. We developed a bioinformatic protocol to analyse ONT sequences taxonomically and functionally and optimized pre-analytic protocols including stool collection and DNA extraction methods to maximize read length. This is a critical parameter for the sequence alignment and classification. Our protocol was evaluated using simulations of metagenomic communities which reflect naturally occuring compositional variations. Next, we validated both protocols using stool samples from a bariatric surgery cohort, sequenced with ONT, Illumina and SOLiD technologies. Results revealed similar diversity and microbial composition profiles. Conclusion: This protocol can be implemented in the clinical or research setting, bringing rapid personalized whole genome profiling of target microbiome species.

A technique for protecting delicate specimens during processing

1989 ◽  
Vol 47 ◽  
pp. 982-983
Author(s):  
R.J. Mount ◽  
R.V. Harrison
Keyword(s):  
Basilar Membrane ◽  
Low Cost ◽  
Organ Of Corti ◽  
Region Of Interest ◽  
Sensory Epithelium ◽  
Physical Damage ◽  
Air Drying ◽  
Specimen Handling ◽  
Two Component

The sensory end organ of the ear, the organ of Corti, rests on a thin basilar membrane which lies between the bone of the central modiolus and the bony wall of the cochlea. In vivo, the organ of Corti is protected by the bony wall which totally surrounds it. In order to examine the sensory epithelium by scanning electron microscopy it is necessary to dissect away the protective bone and expose the region of interest (Fig. 1). This leaves the fragile organ of Corti susceptible to physical damage during subsequent handling. In our laboratory cochlear specimens, after dissection, are routinely prepared by the O-T- O-T-O technique, critical point dried and then lightly sputter coated with gold. This processing involves considerable specimen handling including several hours on a rotator during which the organ of Corti is at risk of being physically damaged. The following procedure uses low cost, readily available materials to hold the specimen during processing ,preventing physical damage while allowing an unhindered exchange of fluids.Following fixation, the cochlea is dehydrated to 70% ethanol then dissected under ethanol to prevent air drying. The holder is prepared by punching a hole in the flexible snap cap of a Wheaton vial with a paper hole punch. A small amount of two component epoxy putty is well mixed then pushed through the hole in the cap. The putty on the inner cap is formed into a “cup” to hold the specimen (Fig. 2), the putty on the outside is smoothed into a “button” to give good attachment even when the cap is flexed during handling (Fig. 3). The cap is submerged in the 70% ethanol, the bone at the base of the cochlea is seated into the cup and the sides of the cup squeezed with forceps to grip it (Fig.4). Several types of epoxy putty have been tried, most are either soluble in ethanol to some degree or do not set in ethanol. The only putty we find successful is “DUROtm MASTERMENDtm Epoxy Extra Strength Ribbon” (Loctite Corp., Cleveland, Ohio), this is a blue and yellow ribbon which is kneaded to form a green putty, it is available at many hardware stores.

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