scholarly journals Long-read viral metagenomics enables capture of abundant and microdiverse viral populations and their niche-defining genomic islands

2018 ◽  
Author(s):  
Joanna Warwick-Dugdale ◽  
Natalie Solonenko ◽  
Karen Moore ◽  
Lauren Chittick ◽  
Ann C. Gregory ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Cost Effective ◽  
Host Community ◽  
Genomic Islands ◽  
Viral Metagenomics ◽  
Viral Gene ◽  
Bioinformatics Pipeline ◽  
Short Read ◽  
Long Read

AbstractMarine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in culturing of important hosts and the lack of a ‘universal’ gene to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution. However, short-read assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Assembly across genomic islands which likely contain niche-defining genes that drive ecological speciation is also challenging. While such populations and features are successfully captured by single-virus genomics and fosmid-based approaches, both techniques require considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative method for improving assembly of viral metagenomics using long read technology. Named ‘VirION’ (Viral, long-read metagenomics via MinION sequencing), our sequencing approach and complementary bioinformatics pipeline (i) increased number and completeness of assembled viral genomes compared to short-read sequencing methods; (ii) captured populations of abundant viruses with high microdiversity missed by short-read methods and (iii) captured more and longer genomic islands than short-read methods. Thus, VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

scholarly journals Long-read viral metagenomics captures abundant and microdiverse viral populations and their niche-defining genomic islands

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6800 ◽  
Author(s):  
Joanna Warwick-Dugdale ◽  
Natalie Solonenko ◽  
Karen Moore ◽  
Lauren Chittick ◽  
Ann C. Gregory ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Host Community ◽  
Genomic Islands ◽  
Viral Metagenomics ◽  
Viral Gene ◽  
Gene Markers ◽  
Short Read ◽  
Viral Genomes ◽  
Long Read

Marine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in host culturing and a lack of reference genomes and ‘universal’ gene markers to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution, but their assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Such features also challenge assembly across genomic islands containing niche-defining genes that drive ecological speciation. These populations and features may be successfully captured by single-virus genomics and fosmid-based approaches, at least in abundant taxa, but at considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative sequencing and informatics workflow to improve viral metagenomic assemblies using short-read and long-read technology. The ‘VirION’ (Viral, long-read metagenomics via MinION sequencing) approach was first validated using mock communities where it was found to be as relatively quantitative as short-read methods and provided significant improvements in recovery of viral genomes. We then then applied VirION to the first metagenome from a natural viral community from the Western English Channel. In comparison to a short-read only approach, VirION: (i) increased number and completeness of assembled viral genomes; (ii) captured abundant, highly microdiverse virus populations, and (iii) captured more and longer genomic islands. Together, these findings suggest that VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

scholarly journals Ultra-low input single tube linked-read library method enables short-read NGS systems to generate highly accurate and economical long-range sequencing information for de novo genome assembly and haplotype phasing

2019 ◽  
Author(s):  
Zhoutao Chen ◽  
Long Pham ◽  
Tsai-Chin Wu ◽  
Guoya Mo ◽  
Yu Xia ◽  
...  
Keyword(s):  
Long Range ◽  
De Novo ◽  
Low Cost ◽  
Cost Effective ◽  
De Novo Genome Assembly ◽  
Short Read ◽  
Single Tube ◽  
Haplotype Phasing ◽  
A Genome ◽  
Long Read

AbstractLong-range sequencing information is required for haplotype phasing, de novo assembly and structural variation detection. Current long-read sequencing technologies can provide valuable long-range information but at a high cost with low accuracy and high DNA input requirement. We have developed a single-tube Transposase Enzyme Linked Long-read Sequencing (TELL-Seq™) technology, which enables a low-cost, high-accuracy and high-throughput short-read next generation sequencer to routinely generate over 100 Kb long-range sequencing information with as little as 0.1 ng input material. In a PCR tube, millions of clonally barcoded beads are used to uniquely barcode long DNA molecules in an open bulk reaction without dilution and compartmentation. The barcode linked reads are used to successfully assemble genomes ranging from microbes to human. These linked-reads also generate mega-base-long phased blocks and provide a cost-effective tool for detecting structural variants in a genome, which are important to identify compound heterozygosity in recessive Mendelian diseases and discover genetic drivers and diagnostic biomarkers in cancers.

scholarly journals High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

2018 ◽  
Author(s):  
Mandeep Singh ◽  
Ghamdan Al-Eryani ◽  
Shaun Carswell ◽  
James M. Ferguson ◽  
James Blackburn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
High Throughput ◽  
Clonal Evolution ◽  
Primary Tumour ◽  
Transcriptome Profiling ◽  
Cost Effective ◽  
Short Read ◽  
Wide Range ◽  
Long Read

AbstractHigh-throughput single-cell RNA-Sequencing is a powerful technique for gene expression profiling of complex and heterogeneous cellular populations such as the immune system. However, these methods only provide short-read sequence from one end of a cDNA template, making them poorly suited to the investigation of gene-regulatory events such as mRNA splicing, adaptive immune responses or somatic genome evolution. To address this challenge, we have developed a method that combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. We use Repertoire And Gene Expression sequencing (RAGE-seq) to accurately characterize full-length T cell (TCR) and B cell (BCR) receptor sequences and transcriptional profiles of more than 7,138 lymphocytes sampled from the primary tumour and draining lymph node of a breast cancer patient. With this method we show that somatic mutation, alternate splicing and clonal evolution of T and B lymphocytes can be tracked across these tissue compartments. Our results demonstrate that RAGE-Seq is an accessible and cost-effective method for high-throughput deep single cell profiling, applicable to a wide range of biological challenges.

scholarly journals VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature

2020 ◽  
Author(s):  
Olivier Zablocki ◽  
Michelle Michelsen ◽  
Marie Burris ◽  
Natalie Solonenko ◽  
Joanna Warwick-Dugdale ◽  
...  
Keyword(s):  
Error Correction ◽  
Previous Method ◽  
Genomic Diversity ◽  
Viral Metagenomics ◽  
Natural Seawater ◽  
Natural Ecosystem ◽  
Gene Markers ◽  
Short Read ◽  
Long Reads ◽  
Long Read

ABSTRACTMicrobes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit— and revealed many critical ecosystem roles for viruses — microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data (‘enhanced’viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow (updated at protocols.io) can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a ‘living document’ to facilitate dissemination of updates to keep pace with the rapid evolution of long read sequencing technology. Taken together, the addition of long-reads uniquely enabled the recovery of 22% of the most abundant viruses—that would have been missed in short-read only assemblies.

scholarly journals A Short-Read Multiplex Sequencing Method for Reliable, Cost-Effective and High-Throughput Genotyping in Large-Scale Studies

2013 ◽  
Vol 35 (2) ◽  
pp. 270-270 ◽  
Author(s):  
Hongzhi Cao ◽  
Yu Wang ◽  
Wei Zhang ◽  
Xianghua Chai ◽  
Xiandong Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Cost Effective ◽  
Short Read ◽  
Sequencing Method ◽  
Multiplex Sequencing

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

2013 ◽  
Vol 42 (4) ◽  
pp. e22-e22 ◽  
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.

High Throughput Electrochemical Method for Contact Optimization in Printed Rectifying Diodes

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.

scholarly journals Detecting SARS-CoV-2 variants with SNP genotyping

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.

scholarly journals Detecting SARS-CoV-2 variants with SNP genotyping

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0243185 ◽  
Author(s):  
Helen Harper ◽  
Amanda Burridge ◽  
Mark Winfield ◽  
Adam Finn ◽  
Andrew Davidson ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Cost Effective ◽  
Snp Genotyping ◽  
Clinical Samples ◽  
Marker Panel ◽  
Qrt Pcr ◽  
Crucial Information ◽  
The Uk ◽  
Genotyping Panel

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.

scholarly journals VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11088
Author(s):  
Olivier Zablocki ◽  
Michelle Michelsen ◽  
Marie Burris ◽  
Natalie Solonenko ◽  
Joanna Warwick-Dugdale ◽  
...  
Keyword(s):  
Error Correction ◽  
Previous Method ◽  
Genomic Diversity ◽  
Viral Metagenomics ◽  
Natural Seawater ◽  
Natural Ecosystem ◽  
Gene Markers ◽  
Short Read ◽  
Long Reads ◽  
Long Read

Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit—and revealed many critical ecosystem roles for viruses—microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data (‘enhanced’ viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity means, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a ‘living document’ to facilitate dissemination of updates to keep pace with the rapid evolution of long-read sequencing technology.

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