Detecting SARS-CoV-2 variants with SNP genotyping

Tracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission but sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations. Genotyping is a rapid, high-throughput and low-cost alternative for screening positive SARS-CoV-2 samples in many settings. We have designed a SNP identification pipeline to identify genetic variation using sequenced SARS-CoV-2 samples. Our pipeline identifies a minimal marker panel that can define distinct genotypes. To evaluate the system, we developed a genotyping panel to detect variants-identified from SARS-CoV-2 sequences surveyed between March and May 2020 and tested this on 50 stored qRT-PCR positive SARS-CoV-2 clinical samples that had been collected across the South West of the UK in April 2020. The 50 samples split into 15 distinct genotypes and there was a 61.9% probability that any two randomly chosen samples from our set of 50 would have a distinct genotype. In a high throughput laboratory, qRT-PCR positive samples pooled into 384-well plates could be screened with a marker panel at a cost of < £1.50 per sample. Our results demonstrate the usefulness of a SNP genotyping panel to provide a rapid, cost-effective, and reliable way to monitor SARS-CoV-2 variants circulating in an outbreak. Our analysis pipeline is publicly available and will allow for marker panels to be updated periodically as viral genotypes arise or disappear from circulation.

Download Full-text

Detecting SARS-CoV-2 variants with SNP genotyping

10.1101/2020.11.18.388140 ◽

2020 ◽

Author(s):

Helen Harper ◽

Amanda J. Burridge ◽

Mark Winfield ◽

Adam Finn ◽

Andrew D. Davidson ◽

...

Keyword(s):

High Throughput ◽

Low Cost ◽

Cost Effective ◽

Snp Genotyping ◽

Clinical Samples ◽

Marker Panel ◽

Qrt Pcr ◽

Crucial Information ◽

The Uk ◽

Genotyping Panel

AbstractTracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission but sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations. Genotyping is a rapid, high-throughput and low-cost alternative for screening positive SARS-CoV-2 samples in many settings. We have designed a SNP identification pipeline to identify genetic variation using sequenced SARS-CoV-2 samples. Our pipeline identifies a minimal marker panel that can define distinct genotypes. To evaluate the system we developed a genotyping panel to detect variants-identified from SARS-CoV-2 sequences surveyed between March and May 2020- and tested this on 50 stored qRT-PCR positive SARS-CoV-2 clinical samples that had been collected across the South West of the UK in April 2020. The 50 samples split into 15 distinct genotypes and there was a 76% probability that any two randomly chosen samples from our set of 50 would have a distinct genotype. In a high throughput laboratory, qRT-PCR positive samples pooled into 384-well plates could be screened with our marker panel at a cost of < £1.50 per sample. Our results demonstrate the usefulness of a SNP genotyping panel to provide a rapid, cost-effective, and reliable way to monitor SARS-CoV-2 variants circulating in an outbreak. Our analysis pipeline is publicly available and will allow for marker panels to be updated periodically as viral genotypes arise or disappear from circulation.

Download Full-text

Technical Innovation in Light Aircraft Aerial Dispersant Application Systems

International Oil Spill Conference Proceedings ◽

10.7901/2169-3358-2003-1-269 ◽

2003 ◽

Vol 2003 (1) ◽

pp. 269-272

Author(s):

David Salt ◽

Roger Stockham ◽

Stuart Byers

Keyword(s):

United Kingdom ◽

Low Cost ◽

Cost Effective ◽

Innovative Technology ◽

Response Strategies ◽

The United Kingdom ◽

Application Systems ◽

Significant Step ◽

Cost Efficient ◽

The Uk

ABSTRACT Recent changes in legislation within the United Kingdom created pressure for change in the response strategies applicable in the UK offshore environment. To meet the new requirements, innovative technology was required which was capable of speedily delivering a payload of approximately one ton of dispersant. To provide a cost efficient solution, a system was developed capable of being mounted on a non-dedicated aircraft, which can be rapidly adapted to meet the response requirements. This paper describes the design criteria for the system and goes on to detail the development, construction and flight testing programme for the dispersant pods. It then goes on to briefly describe the operational response system which has been established to provide a response for the offshore operators in the United Kingdom Continental Shelf (UKCS). The development represents a significant step forward in providing a low cost, effective solution to changing response requirements using innovative engineering solutions, allowing for potential application in other parts of the world.

Download Full-text

Genetic Analysis of colorectal carcinoma using high throughput SNP genotyping technique within the population of Jammu and Kashmir

10.21203/rs.3.rs-199948/v1 ◽

2021 ◽

Author(s):

Bhanu Sharma ◽

Shabab Angurana ◽

Amrita Bhat ◽

Sonali Verma ◽

Divya Bakshi ◽

...

Keyword(s):

Colorectal Cancer ◽

High Throughput ◽

Cost Effective ◽

Snp Genotyping ◽

Colorectal Carcinogenesis ◽

P Value ◽

Jammu And Kashmir ◽

Cost Effective Method ◽

Underlying Causes ◽

Multiple Samples

Abstract Background SNP genotyping has become increasingly more common place to understand the genetic basis of complex diseases like cancer. SNP-genotyping through massARRAY is a cost-effective method to quantitatively analyse the variation of gene expression in multiple samples, making it a potential tool to identify the underlying causes of colorectal carcinogenesis. Methods In the present study, SNP genotyping was carried out using Agena mass ARRAY, which is a cost-effective, robust, and sensitive method to analyse multiple SNPs simultaneously. We analysed 7 genes in 492 samples (100 cases and 392 controls) associated with CRC within the population of Jammu and Kashmir. These SNPs were selected based on their association with multiple cancers in literature. Results This is the first study to explore these SNPs with colorectal cancer within the J&K population.7 SNPs with a call rate of 90% were selected for the study. Out of these, one SNP i.e. rs2229080 of DCC was found to be significantly associated with the current study and 6 were non-significantly associated with CRC within the studied population. The allelic OR observed for the variant rs2229080 of DCC was 1.5 (1.1–2.3 at 95% CI), p value = 0.02. Conclusion This is the first study to find the relation of Genetic variants with the colorectal cancer within the studied population using high throughput mass ARRAY technology. It is further anticipated that the variants should be evaluated in other population groups that may aid in understanding the genetic complexity and bridge the missing heritability.

Download Full-text

High-throughput, cost-effective verification of structural DNA assembly

Nucleic Acids Research ◽

10.1093/nar/gkt1088 ◽

2013 ◽

Vol 42 (4) ◽

pp. e22-e22 ◽

Cited By ~ 14

Author(s):

Yandi Dharmadi ◽

Kedar Patel ◽

Elaine Shapland ◽

Daniel Hollis ◽

Todd Slaby ◽

...

Keyword(s):

High Throughput ◽

Low Cost ◽

Rolling Circle Amplification ◽

Cost Effective ◽

Building Blocks ◽

Pathway Engineering ◽

Dna Assembly ◽

Rolling Circle ◽

Enzyme Screening ◽

First Time

Abstract DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (∼1 kb), pathway engineering (∼10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.

Download Full-text

High Throughput Electrochemical Method for Contact Optimization in Printed Rectifying Diodes

MRS Proceedings ◽

10.1557/opl.2014.386 ◽

2014 ◽

Vol 1628 ◽

Author(s):

Petri S. Heljo ◽

Himadri S. Majumdar ◽

Donald Lupo

Keyword(s):

High Throughput ◽

Low Cost ◽

Cost Effective ◽

Anodic Oxide ◽

Oxide Thickness ◽

Half Wave ◽

Current Voltage ◽

Organic Devices ◽

Anodic Oxidation Method ◽

Semiconductor Contact

ABSTRACTWe report a low cost and high throughput electrochemical anodic oxidation method to enhance the metal-semiconductor contact between a silver electrode and an organic semiconductor in a rectifying diode application. The oxidized layer enhances the contact properties, leading to better device performance. Three different anodic oxide thicknesses were used in the study. Current-voltage and AC rectification measurements were used to characterize the printed devices. The DC output voltage of the half-wave rectifier increased consistently as a function of the oxide thickness. This procedure points toward a cost-effective way to optimize printed organic devices.

Download Full-text

An Acoustic Sensor for Combined Sewer Overflow (CSO) Screen Condition Monitoring in a Drainage Infrastructure

Sensors ◽

10.3390/s21020404 ◽

2021 ◽

Vol 21 (2) ◽

pp. 404

Author(s):

Chan H. See ◽

Kirill V. Horoshenkov ◽

M. Tareq Bin Ali ◽

Simon J. Tait

Keyword(s):

Low Cost ◽

Cost Effective ◽

Acoustic Sensor ◽

Combined Sewer Overflow ◽

Regulatory Requirement ◽

Run Off ◽

Combined Sewer ◽

Sewer Overflow ◽

The Uk ◽

Screen Condition

Combined sewer overflow structures (CSO) play an important role in sewer networks. When the local capacity of a sewer system is exceeded during intense rainfall events, they act as a “safety valve” and discharge excess rainfall run-off and wastewater directly to a natural receiving water body, thus preventing widespread urban flooding. There is a regulatory requirement that solids in CSO spills must be small and their amount strictly controlled. Therefore, a vast majority of CSOs in the UK contain screens. This paper presents the results of a feasibility study of using low-cost, low-energy acoustic sensors to remotely assess the condition of CSO screens to move to cost-effective reactive maintenance visits. In situ trials were carried out in several CSOs to evaluate the performance of the acoustic sensor under realistic screen and flow conditions. The results demonstrate that the system is robust within ±2.5% to work successfully in a live CSO environment. The observed changes in the screen condition resulted in 8–39% changes in the values of the coefficient in the proposed acoustic model. These changes are detectable and consistent with observed screen and hydraulic data. This study suggested that acoustic-based sensing can effectively monitor the CSO screen blockage conditions and hence reduce the risk of non-compliant CSO spills.

Download Full-text

Physical mapping and InDel marker development for the restorer gene Rf2 in cytoplasmic male sterile CMS-D8 cotton

BMC Genomics ◽

10.1186/s12864-020-07342-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Juanjuan Feng ◽

Xuexian Zhang ◽

Meng Zhang ◽

Liping Guo ◽

Tingxiang Qi ◽

...

Keyword(s):

High Throughput ◽

Physical Mapping ◽

Snp Genotyping ◽

Restorer Line ◽

Male Sterile ◽

Cytoplasmic Male Sterile ◽

Qrt Pcr ◽

Indel Markers ◽

Selection Of ◽

Candidate Interval

Abstract Background Cytoplasmic male sterile (CMS) with cytoplasm from Gossypium Trilobum (D8) fails to produce functional pollen. It is useful for commercial hybrid cotton seed production. The restore line of CMS-D8 containing Rf2 gene can restore the fertility of the corresponding sterile line. This study combined the whole genome resequencing bulked segregant analysis (BSA) with high-throughput SNP genotyping to accelerate the physical mapping of Rf2 locus in CMS-D8 cotton. Methods The fertility of backcross population ((sterile line×restorer line)×maintainer line) comprising of 1623 individuals was investigated in the field. The fertile pool (100 plants with fertile phenotypes, F-pool) and the sterile pool (100 plants with sterile phenotypes, S-pool) were constructed for BSA resequencing. The selection of 24 single nucleotide polymorphisms (SNP) through high-throughput genotyping and the development insertion and deletion (InDel) markers were conducted to narrow down the candidate interval. The pentapeptide repeat (PPR) family genes and upregulated genes in restore line in the candidate interval were analysed by qRT-PCR. Results The fertility investigation results showed that fertile and sterile separation ratio was consistent with 1:1. BSA resequencing technology, high-throughput SNP genotyping, and InDel markers were used to identify Rf2 locus on candidate interval of 1.48 Mb on chromosome D05. Furthermore, it was quantified in this experiment that InDel markers co-segregated with Rf2 enhanced the selection of the restorer line. The qRT-PCR analysis revealed PPR family gene Gh_D05G3391 located in candidate interval had significantly lower expression than sterile and maintainer lines. In addition, utilization of anther RNA-Seq data of CMS-D8 identified that the expression level of Gh_D05G3374 encoding NB-ARC domain-containing disease resistance protein in restorer lines was significantly higher than that in sterile and maintainer lines. Conclusions This study not only enabled us to precisely locate the restore gene Rf2 but also evaluated the utilization of InDel markers for marker assisted selection in the CMS-D8 Rf2 cotton breeding line. The results of this study provide an important foundation for further studies on the mapping and cloning of restorer genes.

Download Full-text

Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses

Animals ◽

10.3390/ani11102976 ◽

2021 ◽

Vol 11 (10) ◽

pp. 2976

Author(s):

Edson Kinimi ◽

Mana Mahapatra ◽

Tebogo Kgotlele ◽

Mariam R. Makange ◽

Chandana Tennakoon ◽

...

Keyword(s):

Low Cost ◽

Viral Evolution ◽

Cost Effective ◽

Nucleotide Identity ◽

Clinical Samples ◽

Depth Information ◽

Peste Des Petits Ruminants ◽

Sequencing Technology ◽

Oxford Nanopore ◽

Complete Genomes

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.

Download Full-text

Genetic Analysis of colorectal carcinoma using high throughput SNP genotyping technique within the population of Jammu and Kashmir.

10.21203/rs.3.rs-199948/v2 ◽

2021 ◽

Author(s):

Bhanu Sharma ◽

Shabab Angurana ◽

Amrita Bhat ◽

Sonali Verma ◽

Divya Bakshi ◽

...

Keyword(s):

Colorectal Cancer ◽

High Throughput ◽

Cost Effective ◽

Snp Genotyping ◽

Colorectal Carcinogenesis ◽

P Value ◽

Jammu And Kashmir ◽

Cost Effective Method ◽

Underlying Causes ◽

Multiple Samples

Abstract Background SNP genotyping has become increasingly more common place to understand the genetic basis of complex diseases like cancer. SNP-genotyping through massARRAY is a cost-effective method to quantitatively analyse the variation of gene expression in multiple samples, making it a potential tool to identify the underlying causes of colorectal carcinogenesis.Methods In the present study, SNP genotyping was carried out using Agena mass ARRAY, which is a cost-effective, robust, and sensitive method to analyse multiple SNPs simultaneously. We analysed 7 genes in 492 samples (100 cases and 392 controls) associated with CRC within the population of Jammu and Kashmir. These SNPs were selected based on their association with multiple cancers in literature.Results This is the first study to explore these SNPs with colorectal cancer within the J&K population.7 SNPs with a call rate of 90% were selected for the study. Out of these, one SNP i.e. rs2229080 of DCC was found to be significantly associated with the current study and 6 were non-significantly associated with CRC within the studied population. The allelic OR observed for the variant rs2229080 of DCC was 1.5 (1.1–2.3 at 95% CI), p value = 0.02.Conclusion This is the first study to find the relation of Genetic variants with the colorectal cancer within the studied population using high throughput mass ARRAY technology. It is further anticipated that the variants should be evaluated in other population groups that may aid in understanding the genetic complexity and bridge the missing heritability.

Download Full-text

Physical mapping and InDel marker development for the restorer gene Rf2 in cytoplasmic male sterile CMS-D8 cotton

10.21203/rs.3.rs-57917/v3 ◽

2021 ◽

Author(s):

Juanjuan Feng ◽

Xuexian Zhang ◽

Meng Zhang ◽

Liping Guo ◽

Tingxiang QI ◽

...

Keyword(s):

High Throughput ◽

Physical Mapping ◽

Snp Genotyping ◽

Restorer Line ◽

Male Sterile ◽

Cytoplasmic Male Sterile ◽

Qrt Pcr ◽

Indel Markers ◽

Selection Of ◽

Candidate Interval

Abstract Background: Cytoplasmic male sterile (CMS) with cytoplasm from Gossypium Trilobum (D8) fails to produce functional pollen. It is useful for commercial hybrid cotton seed production. The restore line of CMS-D8 containing Rf2 gene can restore the fertility of the corresponding sterile line. This study combined the whole genome resequencing bulked segregant analysis (BSA) with high-throughput SNP genotyping to accelerate the physical mapping of Rf2 locus in CMS-D8 cotton. Methods: The fertility of backcross population ((sterile line×restorer line)×maintainer line) comprising of 1623 individuals was investigated in the field. The fertile pool (100 plants with fertile phenotypes, F-pool) and the sterile pool (100 plants with sterile phenotypes, S-pool) were constructed for BSA resequencing. The selection of 24 single nucleotide polymorphisms (SNP) through high-throughput genotyping and the development insertion and deletion (InDel) markers were conducted to narrow down the candidate interval. The pentapeptide repeat (PPR) family genes and upregulated genes in restore line in the candidate interval were analysed by qRT-PCR. Results: The fertility investigation results showed that fertile and sterile separation ratio was consistent with 1:1. BSA resequencing technology, high-throughput SNP genotyping, and InDel markers were used to identify Rf2 locus on candidate interval of 1.48 Mb on Chromosome D05. Furthermore, it was quantified in this experiment that InDel markers co-segregated with Rf2 enhanced the selection of the restorer line. The qRT-PCR analysis revealed PPR family gene Gh_D05G3391 located in candidate interval had significantly lower expression than sterile and maintainer lines. In addition, utilization of anther RNA-Seq data of CMS-D8 identified that the expression level of Gh_D05G3374 encoding NB-ARC domain-containing disease resistance protein in restorer lines was significantly higher than that in sterile and maintainer lines. Conclusions: This study not only enabled us to precisely locate the restore gene Rf2 but also evaluated the utilization of InDel markers for marker assisted selection in the CMS-D8 Rf2 cotton breeding line. The results of this study provide an important foundation for further studies on the mapping and cloning of restorer genes.

Download Full-text