A streamlined protocol for high-throughput amplification-based analysis of DNA samples via nanopore sequencing (based on the 96-well PCR barcoding kit) v1

2021 ◽  
Author(s):  
Jordan P Cuff ◽  
HooperAS not provided ◽  
Shrinivas Nivrutti Dighe ◽  
Angela Marchbank ◽  
Peter Kille
Keyword(s):  
High Throughput ◽  
Genomic Dna ◽  
Dna Amplification ◽  
Cost Effective ◽  
Nanopore Sequencing ◽  
Optimal Method ◽  
Adapter Ligation ◽  
Large Numbers ◽  
Oxford Nanopore

Nanopore sequencing facilitates the rapid and cost-effective sequencing of long fragment DNA for a massive range of applications. When looking to holistically analyse low-yield DNA samples using nanopore sequencing, the optimal method is likely to involve the PCR Barcoding Kit. This effectively involves blunt end ligation of priming sites onto all extant DNA for holistic amplification to achieve yields suitable for nanopore sequencing. The currently available kits from nanopore facilitate the multiplexing of 96 samples in one sequencing run using this method, but the reagent costs are inherently multiplicative. This protocol is designed to streamline (in terms of cost, reagents and time) the process of sequencing up to 96 samples of genomic DNA through nanopore sequencing. This protocol is best applied to large numbers of samples (up to 96). For smaller numbers of samples, consider the smaller "PCR Barcoding" kits provided by nanopore which similarly achieve holistic DNA amplification and sequencing, but without the need for additional adapter ligation. The protocol is best suited to samples with low DNA yields (100 ng input is recommended). If you can input 1000 ng of DNA from each of your samples, consider using the 96-well Ligation Kit from Oxford Nanopore which can similarly be streamlined in terms of cost and time, but avoids the amplification step.

scholarly journals Detection of single nucleotide and copy number variants in the Fabry disease-associated GLA gene using nanopore sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Albina Nowak ◽  
Omer Murik ◽  
Tzvia Mann ◽  
David A. Zeevi ◽  
Gheona Altarescu
Keyword(s):  
Fabry Disease ◽  
Copy Number ◽  
New Technology ◽  
Copy Number Variants ◽  
Cost Effective ◽  
Nanopore Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sanger Sequence ◽  
Gla Gene

AbstractMore than 900 variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence. We aimed to design and validate a method for sequencing the GLA gene using long-read Oxford Nanopore sequencing technology. Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long-read sequencing of a 13 kb PCR amplicon. We used minimap2 to align the long-read data and Nanopolish and Sniffles to call variants. All the variants detected by Sanger (including a deep intronic variant) were also detected by long-read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology. Our long-read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.

scholarly journals Detection of Single Nucleotide and Copy Number Variants in the Fabry Disease-associated GLA Gene Using Nanopore Sequencing

2021 ◽  
Author(s):  
Albina Nowak ◽  
Omer Murik ◽  
Tzvia Mann ◽  
David A. Zeevi ◽  
Gheona Altarescu
Keyword(s):  
Fabry Disease ◽  
Copy Number ◽  
New Technology ◽  
Copy Number Variants ◽  
Cost Effective ◽  
Nanopore Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sanger Sequence ◽  
Gla Gene

Abstract Introduction: More than one thousand variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence.Aims: We aimed to design and validate a method for sequencing the GLA gene using long read Oxford Nanopore sequencing technology.Methods: Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long read sequencing of a 13kb PCR amplicon. We used minimap2 to align the long read data and Nanopolish and Sniffles to call variants.Results: All the variants detected by Sanger (including a deep intronic variant) were also detected by long read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology.Conclusions: Our long read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.

scholarly journals Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy

2020 ◽  
Vol 8 (1) ◽  
pp. e000299
Author(s):  
Ping Zhang ◽  
Devika Ganesamoorthy ◽  
Son Hoang Nguyen ◽  
Raymond Au ◽  
Lachlan J Coin ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Cost Effective ◽  
Inverse Pcr ◽  
Nanopore Sequencing ◽  
Next Generation ◽  
Short Read ◽  
Vector Integration ◽  
Integration Sites ◽  
Generation Sequencing

BackgroundAnalysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and large batch runs.MethodsWe used nanopore sequencing to analyze the vector integration sites of T cells transduced by the gammaretroviral vector, SFG.iCasp9.2A.ΔCD19. DNA from oligoclonal cell lines and polyclonal clinical samples were restriction enzyme digested with two 6-cutters,NcoIandBspHI; and the flanking genomic DNA amplified by inverse PCR or cassette ligation PCR. Following nested PCR and barcoding, the amplicons were sequenced on the Oxford Nanopore platform. Reads were filtered for quality, trimmed, and aligned. Custom tool was developed to cluster reads and merge overlapping clusters.ResultsBoth inverse PCR and cassette ligation PCR could successfully amplify flanking genomic DNA, with cassette ligation PCR showing less bias. The 4.8 million raw reads were grouped into 12,186 clusters and 6410 clones. The 3′long terminal repeat (LTR)-genome junction could be resolved within a 5-nucleotide span for a majority of clusters and within one nucleotide span for clusters with ≥5 reads. The chromosomal distributions of the insertional sites and their predilection for regions proximate to transcription start sites were consistent with previous reports for gammaretroviral vector integrants as analyzed by short-read next-generation sequencing.ConclusionOur study shows that it is feasible to use nanopore sequencing to map polyclonal vector integration sites. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis. Further refinement is required to reduce amplification bias and improve single nucleotide resolution.

scholarly journals Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology

2021 ◽  
Vol 9 (12) ◽  
pp. 2598
Author(s):  
Anton Pembaur ◽  
Erwan Sallard ◽  
Patrick Philipp Weil ◽  
Jennifer Ortelt ◽  
Parviz Ahmad-Nejad ◽  
...  
Keyword(s):  
Point Of Care ◽  
Cost Effective ◽  
Nanopore Sequencing ◽  
Bioinformatics Pipeline ◽  
Sequencing Technologies ◽  
Copy Numbers ◽  
Oxford Nanopore ◽  
Novel Variants ◽  
Primer Sets ◽  
Working Day

The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the ‘midnight’ 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 106 were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage.

scholarly journals Sequencing ultra-long DNA molecules with the Oxford Nanopore MinION

2015 ◽  
Author(s):  
John M Urban ◽  
Jacob Bliss ◽  
Charles E Lawrence ◽  
Susan A Gerbi
Keyword(s):  
Cost Effective ◽  
Nanopore Sequencing ◽  
Dna Fragments ◽  
Dna Molecules ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

Oxford Nanopore Technologies' nanopore sequencing device, the MinION, holds the promise of sequencing ultra-long DNA fragments >100kb. An obstacle to realizing this promise is delivering ultra-long DNA molecules to the nanopores. We present our progress in developing cost-effective ways to overcome this obstacle and our resulting MinION data, including multiple reads >100kb.

scholarly journals Determining Exon Connectivity in Complex mRNAs by Nanopore Sequencing

2015 ◽  
Author(s):  
Mohan Bolisetty ◽  
Gopinath Rajadinakaran ◽  
Brenton Graveley
Keyword(s):  
Rna Sequencing ◽  
High Throughput ◽  
Full Length ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Important Method ◽  
Oxford Nanopore ◽  
Multiple Alternative

Though powerful, short-read high throughput RNA sequencing is limited in its ability to directly measure exon connectivity in mRNAs containing multiple alternative exons located farther apart than the maximum read lengths. Here, we use the Oxford Nanopore MinION™ sequencer to identify 7,899 ‘full-length’ isoforms expressed from four Drosophila genes, Dscam1, MRP, Mhc, and Rdl. These results demonstrate that nanopore sequencing can be used to deconvolute individual isoforms and that it has the potential to be an important method for comprehensive transcriptome characterization.

scholarly journals Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing

2021 ◽  
Vol 22 (18) ◽  
pp. 9723
Author(s):  
Paul A. P. Atkins ◽  
Maria Elena S. Gamo ◽  
Daniel F. Voytas
Keyword(s):  
Plant Regeneration ◽  
Genome Editing ◽  
Gene Targeting ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Nanopore Sequencing ◽  
Bioinformatic Pipeline ◽  
Oxford Nanopore ◽  
Mechanistic Basis ◽  
Ngs Pipelines

The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such as ddPCR. In plants, removal of excess donor is time and resource intensive, often requiring plant regeneration and weeks to months of effort. Here, we utilized Oxford Nanopore Amplicon Sequencing (ONAS) to bypass the limitations imposed by donor molecules with 1 kb of homology to the target and dissected GT outcomes at three loci in Nicotiana benthamia leaves. We developed a novel bioinformatic pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to reduce the effect of ONAS error on amplicon analysis and captured tens of thousands of somatic plant GT events. Additionally, PANGEA allowed us to collect thousands of GT conversion tracts 5 days after reagent delivery with no selection, revealing that most events utilized tracts less than 100 bp in length when incorporating an 18 bp or 3 bp insertion. These data demonstrate the usefulness of ONAS and PANGEA for plant GT analysis and provide a mechanistic basis for future plant GT optimization.

scholarly journals Diagnostic accuracy of Loop mediated isothermal amplification coupled to Nanopore sequencing for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations

2020 ◽  
Author(s):  
Anetta Ptasinska ◽  
Celina Whalley ◽  
Andrew Bosworth ◽  
Charlie Poxon ◽  
Clare Bryer ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
High Throughput ◽  
Health Care Workers ◽  
Isothermal Amplification ◽  
Nanopore Sequencing ◽  
Sequencing Technology ◽  
Loop Mediated Isothermal Amplification ◽  
Oxford Nanopore ◽  
Care Workers ◽  
Diagnostic Sensitivity And Specificity

Introduction: Rapid, high throughput diagnostics are a valuable tool, allowing the detection of SARS-CoV-2 in populations, in order to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as reverse transcriptase quantitative PCR (RTqPCR), particularly throughout the first months of the pandemic. We investigated the use of LamPORE, where loop mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS CoV 2 in symptomatic and asymptomatic populations. Methods: In an asymptomatic prospective cohort; health care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self swabbed with nasopharyngeal swabs weekly for three weeks and supplied a saliva specimen daily. These samples were tested for SARS CoV 2 RNA using the Oxford Nanopore LamPORE system and a reference RTqPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza like illness from March 2020 to June 2020, were similarly tested from nasopharyngeal swabs. Results: In the asymptomatic cohort a total of 1200 participants supplied 23,427 samples (3,966 swab, 19,461 saliva) over a three-week period. The incidence of SARS CoV 2 was 0.95% using LamPORE. Diagnostic sensitivity and specificity was > 99.5% in both swab and saliva asymptomatic samples as compared to the reference RTqPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. Conclusions: LamPORE is a highly accurate methodology for the detection of SARS CoV 2 in both the symptomatic and asymptomatic population settings and can be used as an alternative to RTqPCR.

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