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.

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 Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

2019 ◽  
Author(s):  
Søren M. Karst ◽  
Ryan M. Ziels ◽  
Rasmus H. Kirkegaard ◽  
Emil A. Sørensen ◽  
Daniel McDonald ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Amplicon Sequencing ◽  
High Accuracy ◽  
Pacbio Sequencing ◽  
Consensus Sequences ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genomic Regions ◽  
Oxford Nanopore Technologies

AbstractHigh-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies or Pacific Biosciences CCS sequencing, yielding high accuracy single-molecule consensus sequences of large genomic regions. Our approach generates amplicon and genomic sequences of >10,000 bp in length with a mean error-rate of 0.0049-0.0006% and chimera rate <0.022%.

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.

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 Extremely Halophilic Biohydrogen Producing Microbial Communities from High-Salinity Soil and Salt Evaporation Pond

Fuels ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 241-252
Author(s):  
Dyah Asri Handayani Taroepratjeka ◽  
Tsuyoshi Imai ◽  
Prapaipid Chairattanamanokorn ◽  
Alissara Reungsang
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
High Salinity ◽  
Amplicon Sequencing ◽  
Spatial Proximity ◽  
Lignocellulosic Waste ◽  
Evaporation Pond ◽  
Operational Taxonomic Units ◽  
Determining Factor

Extreme halophiles offer the advantage to save on the costs of sterilization and water for biohydrogen production from lignocellulosic waste after the pretreatment process with their ability to withstand extreme salt concentrations. This study identifies the dominant hydrogen-producing genera and species among the acclimatized, extremely halotolerant microbial communities taken from two salt-damaged soil locations in Khon Kaen and one location from the salt evaporation pond in Samut Sakhon, Thailand. The microbial communities’ V3–V4 regions of 16srRNA were analyzed using high-throughput amplicon sequencing. A total of 345 operational taxonomic units were obtained and the high-throughput sequencing confirmed that Firmicutes was the dominant phyla of the three communities. Halanaerobium fermentans and Halanaerobacter lacunarum were the dominant hydrogen-producing species of the communities. Spatial proximity was not found to be a determining factor for similarities between these extremely halophilic microbial communities. Through the study of the microbial communities, strategies can be developed to increase biohydrogen molar yield.

scholarly journals Genome and transcriptome of a pathogenic yeast, Candida nivariensis

2021 ◽  
Author(s):  
Yunfan Fan ◽  
Andrew N Gale ◽  
Anna Bailey ◽  
Kali Barnes ◽  
Kiersten Colotti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Nanopore Sequencing ◽  
Cdna Sequencing ◽  
Pathogenic Yeast ◽  
Mitochondrial Sequence ◽  
Dna And Rna ◽  
Oxford Nanopore ◽  
Candida Nivariensis ◽  
Oxford Nanopore Technologies

Abstract We present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies (ONT) and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.

scholarly journals Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Francesca De Filippis ◽  
Manolo Laiola ◽  
Giuseppe Blaiotta ◽  
Danilo Ercolini
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Biological Samples ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Fungal Species ◽  
Fungal Communities ◽  
Its2 Region ◽  
Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

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