scholarly journals COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance

2021 ◽  
Author(s):  
Michele Simonetti ◽  
Ning Zhang ◽  
Luuk Harbers ◽  
Maria Grazia Milia ◽  
Silvia Brossa ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Cost Effective ◽  
Mass Scale ◽  
Influenza Viruses ◽  
Library Preparation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Effective Manner ◽  
Vaccination Campaigns ◽  
Standard Library

Abstract While mass-scale vaccination campaigns are ongoing worldwide, genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to monitor the emergence and global spread of viral variants of concern (VOC). Here, we present a streamlined workflow—COVseq—which can be used to generate highly multiplexed sequencing libraries compatible with Illumina platforms from hundreds of SARS-CoV-2 samples in parallel, in a rapid and cost-effective manner. We benchmarked COVseq against a standard library preparation method (NEBNext) on 29 SARS-CoV-2 positive samples, reaching 95.4% of concordance between single-nucleotide variants detected by both methods. Application of COVseq to 245 additional SARS-CoV-2 positive samples demonstrated the ability of the method to reliably detect emergent VOC as well as its compatibility with downstream phylogenetic analyses. A cost analysis showed that COVseq could be used to sequence thousands of samples at less than 15 USD per sample, including library preparation and sequencing costs. We conclude that COVseq is a versatile and scalable method that is immediately applicable for SARS-CoV-2 genomic surveillance and easily adaptable to other pathogens such as influenza viruses.

scholarly journals COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance

2021 ◽  
Author(s):  
Michele Simonetti ◽  
Ning Zhang ◽  
Luuk Harbers ◽  
Maria Grazia Milia ◽  
Silvia Brossa ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Cost Effective ◽  
Mass Scale ◽  
Influenza Viruses ◽  
Library Preparation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Effective Manner ◽  
Vaccination Campaigns ◽  
Standard Library

Abstract While mass-scale vaccination campaigns are ongoing worldwide, genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to monitor the emergence and global spread of viral variants of concern (VOC). Here, we present a streamlined workflow—COVseq—which can be used to generate highly multiplexed sequencing libraries compatible with Illumina platforms from hundreds of SARS-CoV-2 samples in parallel, in a rapid and cost-effective manner. We benchmarked COVseq against a standard library preparation method (NEBNext) on 29 SARS-CoV-2 positive samples, reaching 95.4% of concordance between single-nucleotide variants detected by both methods. Application of COVseq to 245 additional SARS-CoV-2 positive samples demonstrated the ability of the method to reliably detect emergent VOC as well as its compatibility with downstream phylogenetic analyses. A cost analysis showed that COVseq could be used to sequence thousands of samples at less than 15 USD per sample, including library preparation and sequencing costs. We conclude that COVseq is a versatile and scalable method that is immediately applicable for SARS-CoV-2 genomic surveillance and easily adaptable to other pathogens such as influenza viruses.

scholarly journals COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michele Simonetti ◽  
Ning Zhang ◽  
Luuk Harbers ◽  
Maria Grazia Milia ◽  
Silvia Brossa ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Cost Effective ◽  
Mass Scale ◽  
Influenza Viruses ◽  
Library Preparation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Effective Manner ◽  
Vaccination Campaigns ◽  
Standard Library

AbstractWhile mass-scale vaccination campaigns are ongoing worldwide, genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to monitor the emergence and global spread of viral variants of concern (VOC). Here, we present a streamlined workflow—COVseq—which can be used to generate highly multiplexed sequencing libraries compatible with Illumina platforms from hundreds of SARS-CoV-2 samples in parallel, in a rapid and cost-effective manner. We benchmark COVseq against a standard library preparation method (NEBNext) on 29 SARS-CoV-2 positive samples, reaching 95.4% of concordance between single-nucleotide variants detected by both methods. Application of COVseq to 245 additional SARS-CoV-2 positive samples demonstrates the ability of the method to reliably detect emergent VOC as well as its compatibility with downstream phylogenetic analyses. A cost analysis shows that COVseq could be used to sequence thousands of samples at less than 15 USD per sample, including library preparation and sequencing costs. We conclude that COVseq is a versatile and scalable method that is immediately applicable for SARS-CoV-2 genomic surveillance and easily adaptable to other pathogens such as influenza viruses.

scholarly journals COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance

2021 ◽  
Author(s):  
Michele Simonetti ◽  
Ning Zhang ◽  
Luuk Harbers ◽  
Maria Grazia Milia ◽  
Thi Nguyen ◽  
...  
Keyword(s):  
Protein A ◽  
Cost Effective ◽  
Mass Scale ◽  
Influenza Viruses ◽  
Spike Protein ◽  
Library Preparation ◽  
Genome Coverage ◽  
Effective Manner ◽  
Geographical Regions ◽  
Different Populations

Abstract Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to monitor the spread and evolution of the virus across different populations, geographical regions and species. Here, we present a streamlined workflow—COVseq—based on the CUTseq method that we previously described, which can be used to generate highly multiplexed sequencing libraries compatible with Illumina platforms, from hundreds of SARS-CoV-2 samples in parallel, in a rapid and cost-effective manner. We validated COVseq on RNA extracted from the supernatant of a SARS-CoV-2 culture as well as from 85 RNA samples from nasopharyngeal swabs, demonstrating the ability of COVseq to achieve near complete genome coverage, including the S region encoding the spike protein. A cost analysis showed that COVseq could be used to sequence thousands of samples per week at less than 10 USD per sample, including library preparation and sequencing costs. COVseq is a versatile and scalable method that can be readily applied for genomic surveillance of the ongoing pandemic and easily adapted to other pathogens such as influenza viruses.

scholarly journals COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance

2021 ◽  
Author(s):  
Michele Simonetti ◽  
Ning Zhang ◽  
Luuk Harbers ◽  
Maria Grazia Milia ◽  
Thi Thu Huong Nguyen ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Protein A ◽  
Cost Effective ◽  
Mass Scale ◽  
Influenza Viruses ◽  
Spike Protein ◽  
Genome Coverage ◽  
Effective Manner ◽  
Geographical Regions ◽  
Different Populations

Abstract Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to monitor the spread and evolution of the virus across different populations, geographical regions and species. Here, we present a streamlined workflow—COVseq—based on the CUTseq method that we previously described, which can be used to generate highly multiplexed sequencing libraries compatible with Illumina platforms, from hundreds of SARS-CoV-2 samples in parallel, in a rapid and cost-effective manner. We validated COVseq on RNA extracted from the supernatant of a SARS-CoV-2 culture as well as from 85 left-over samples from nasopharyngeal swabs, demonstrating the ability of COVseq to achieve almost complete genome coverage, including the S region encoding the spike protein. A cost analysis showed that COVseq could be used to sequence thousands of samples per week at less than 20 USD per sample. COVseq is a versatile and scalable method that can be readily applied for genomic surveillance of the ongoing pandemic and easily adapted to other pathogens such as influenza viruses.

scholarly journals Strand-wise and bait-assisted assembly of nearly-full rrn operons applied to assess species engraftment after faecal microbiota transplantation

2020 ◽  
Author(s):  
Alfonso Benítez-Páez ◽  
Annick V. Hartstra ◽  
Max Nieuwdorp ◽  
Yolanda Sanz
Keyword(s):  
Gut Microbiota ◽  
Bacterial Species ◽  
Cost Effective ◽  
Strain Level ◽  
Level Variation ◽  
Faecal Microbiota ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Faecal Microbiota Transplantation ◽  
Effective Manner

AbstractBackgroundEffective methodologies to accurately identify members of the gut microbiota at the species and strain levels are necessary to unveiling more specific and detailed host-microbe interactions and associations with health and disease.MethodsMinION™ MkIb nanopore-based device and the R9.5 flowcell chemistry were used to sequence and assemble dozens of rrn regions (16S-ITS-23S) derived from the most prevalent bacterial species in the human gut microbiota. As a method proof-of-concept to disclose further strain-level variation, we performed a complementary analysis in a subset of samples derived from an faecal microbiota transplantation (FMT) trial aiming amelioration of glucose and lipid metabolism in overweight subjects with metabolic syndrome.ResultsThe resulting updated rrn database, the data processing pipeline, and the precise control of covariates (sequencing run, sex, age, BMI, donor) were pivotal to accurately estimate the changes in gut microbial species abundance in the recipients after FMT. Furthermore, the rrn methodology described here demonstrated the ability to detect strain-level variation, critical to evaluate the transference of bacteria from donors to recipients as a consequence of the FMT. At this regard, we showed that our FMT trial successfully induced donors’ strain engraftment of e.g. Parabacteroides merdae species in recipients by mapping and assessing their associated single nucleotide variants (SNV).ConclusionsWe developed a methodology that enables the identification of microbiota at species- and strain-level in a cost-effective manner. Despite its error-prone nature and its modest per-base accuracy, the nanopore data showed to have enough quality to estimate single-nucleotide variation. This methodology and data analysis represents a cost-effective manner to trace genetic variability needed for better understanding the health effects of the human microbiome.Trial registrationThe study was prospectively registered at the Dutch Trial registry - NTR4488 (https://www.trialregister.nl/trial/4488).

scholarly journals Highly multiplexed, fast and accurate nanopore sequencing for verification of synthetic DNA constructs and sequence libraries

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrew Currin ◽  
Neil Swainston ◽  
Mark S Dunstan ◽  
Adrian J Jervis ◽  
Paul Mulherin ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Sequencing ◽  
Cost Effective ◽  
Polymorphism Analysis ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Synthetic Dna ◽  
Design Build ◽  
Hardware Costs

Abstract Synthetic biology utilizes the Design–Build–Test–Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from Escherichia coli colonies to analyzed data). Given our procedure’s low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis.

scholarly journals A genotype network reveals homoplastic cycles of convergent evolution in influenza A (H3N2) haemagglutinin

2014 ◽  
Vol 281 (1786) ◽  
pp. 20132763 ◽  
Author(s):  
Andreas Wagner
Keyword(s):  
Network Analysis ◽  
Influenza A ◽  
Evolutionary Dynamics ◽  
Phylogenetic Analyses ◽  
Influenza Viruses ◽  
Spatial Proximity ◽  
Evolutionary Constraints ◽  
Single Nucleotide ◽  
Time Resolved ◽  
Organizational Patterns

Networks of evolving genotypes can be constructed from the worldwide time-resolved genotyping of pathogens like influenza viruses. Such genotype networks are graphs where neighbouring vertices (viral strains) differ in a single nucleotide or amino acid. A rich trove of network analysis methods can help understand the evolutionary dynamics reflected in the structure of these networks. Here, I analyse a genotype network comprising hundreds of influenza A (H3N2) haemagglutinin genes. The network is rife with cycles that reflect non-random parallel or convergent (homoplastic) evolution. These cycles also show patterns of sequence change characteristic for strong and local evolutionary constraints, positive selection and mutation-limited evolution. Such cycles would not be visible on a phylogenetic tree, illustrating that genotype network analysis can complement phylogenetic analyses. The network also shows a distinct modular or community structure that reflects temporal more than spatial proximity of viral strains, where lowly connected bridge strains connect different modules. These and other organizational patterns illustrate that genotype networks can help us study evolution in action at an unprecedented level of resolution.

scholarly journals Genome-Wide Development and Validation of Cost-Effective KASP Marker Assays for Genetic Dissection of Heat Stress Tolerance in Maize

2020 ◽  
Vol 21 (19) ◽  
pp. 7386
Author(s):  
Ashok Babadev Jagtap ◽  
Yogesh Vikal ◽  
Gurmukh Singh Johal
Keyword(s):  
Heat Stress ◽  
Stress Tolerance ◽  
Cost Effective ◽  
Genetic Dissection ◽  
Rna Seq ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Heat Stress Tolerance ◽  
Kasp Markers

Maize is the third most important cereal crop worldwide. However, its production is vulnerable to heat stress, which is expected to become more and more severe in coming years. Germplasm resilient to heat stress has been identified, but its underlying genetic basis remains poorly understood. Genomic mapping technologies can fill the void, provided robust markers are available to tease apart the genotype-phenotype relationship. In the present investigation, we used data from an RNA-seq experiment to identify single nucleotide polymorphisms (SNPs) between two contrasting lines, LM11 and CML25, sensitive and tolerant to heat stress, respectively. The libraries for RNA-seq were made following heat stress treatment from three separate tissues/organs, comprising the top leaf, ovule, and pollen, all of which are highly vulnerable to damage by heat stress. The single nucleotide variants (SNVs) calling used STAR mapper and GATK caller pipelines in a combined approach to identify highly accurate SNPs between the two lines. A total of 554,423, 410,698, and 596,868 SNVs were discovered between LM11 and CML25 after comparing the transcript sequence reads from the leaf, pollen, and ovule libraries, respectively. Hundreds of these SNPs were then selected to develop into genome-wide Kompetitive Allele-Specific PCR (KASP) markers, which were validated to be robust with a successful SNP conversion rate of 71%. Subsequently, these KASP markers were used to effectively genotype an F2 mapping population derived from a cross of LM11 and CML25. Being highly cost-effective, these KASP markers provide a reliable molecular marker toolkit to not only facilitate the genetic dissection of the trait of heat stress tolerance but also to accelerate the breeding of heat-resilient maize by marker-assisted selection (MAS).

scholarly journals Development of a real-time PCR assay to detect the single nucleotide polymorphism causing Warmblood Fragile Foal Syndrome

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259316
Author(s):  
Sharon Flanagan ◽  
Áine Rowe ◽  
Vivienne Duggan ◽  
Erin Markle ◽  
Maureen O’Brien ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Genetic Mutation ◽  
Wild Type ◽  
High Throughput Analysis ◽  
Single Nucleotide ◽  
Cornell University ◽  
Single Nucleotide Change ◽  
Effective Manner

Warmblood Fragile Foal syndrome (WFFS) is an autosomal recessive condition that affects the maturation of collagen in affected foals. Foals affected with the disease typically die or are euthanised shortly after birth. WFFS is caused by a single nucleotide change at position 2032 of the equine PLOD1 gene, causing an impairment of the wild-type enzyme. A commercial test for the causative genetic mutation is currently available from companies operating under licence from Cornell University but it has limitations. This test requires amplification of a region of the PLOD1 gene encompassing the site of interest, followed by Sanger sequencing of that region and computational analysis. We describe here the development of an alternative, real-time PCR based assay that rapidly and reliably differentiates between the wild-type and WFFS associated nucleotides without the need for sequencing, thus increasing the potential for high throughput analysis of large numbers of samples in a cost-effective manner.

High-throughput genotyping assay for the large-scale genetic characterization ofCryptosporidiumparasites from human and bovine samples

Parasitology ◽  
2013 ◽  
Vol 141 (4) ◽  
pp. 491-500 ◽  
Author(s):  
J. L. ABAL-FABEIRO ◽  
X. MASIDE ◽  
J. LLOVO ◽  
X. BELLO ◽  
M. TORRES ◽  
...  
Keyword(s):  
High Throughput ◽  
Sanger Sequencing ◽  
Large Scale ◽  
Genetic Characterization ◽  
Low Cost ◽  
Cost Effective ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Identification Of Species

SUMMARYThe epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of commongp60subtypes of fourCryptosporidiumspecies that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) andgp60subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four differentCryptosporidiumspecies (C. hominis, C. parvum, C. meleagridisandC. felis) were detected in humans; the most common ones wereC. hominissubtype Ib, andC. parvumIIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.

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