scholarly journals A rapid and accurate MinION-based workflow for tracking species biodiversity in the field

2019 ◽  
Author(s):  
Simone Maestri ◽  
Emanuela Cosentino ◽  
Marta Paterno ◽  
Hendrik Freitag ◽  
Jhoana M. Garces ◽  
...  
Keyword(s):  
De Novo ◽  
Field Conditions ◽  
Dna Barcodes ◽  
Consensus Sequences ◽  
Oxford Nanopore ◽  
Assembly Pipeline ◽  
Adverse Environmental Conditions ◽  
User Friendly ◽  
Oxford Nanopore Technologies ◽  
Standard Molecular Biology

AbstractGenetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION platform. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure reliable and robust performance under suboptimal field conditions without increasing costs. Here we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 500 reads per sample. ONTrack-derived consensus barcodes have high accuracy, ranging from 99,8% to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.

scholarly journals A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 468 ◽  
Author(s):  
Maestri ◽  
Cosentino ◽  
Paterno ◽  
Freitag ◽  
Garces ◽  
...  
Keyword(s):  
De Novo ◽  
Field Conditions ◽  
Dna Barcodes ◽  
Consensus Sequences ◽  
Oxford Nanopore ◽  
Assembly Pipeline ◽  
Adverse Environmental Conditions ◽  
User Friendly ◽  
Oxford Nanopore Technologies ◽  
Standard Molecular Biology

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.

scholarly journals Complete Circular Genome Sequences of Brachyspira hyodysenteriae Isolates of the Four Different Sequence Types Causing Swine Dysentery in Switzerland

2021 ◽  
Vol 10 (39) ◽  
Author(s):  
Ana B. García-Martín ◽  
Sarah Schmitt ◽  
Friederike Zeeh ◽  
Vincent Perreten
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Hybrid Assembly ◽  
Swine Dysentery ◽  
Content Type ◽  
Brachyspira Hyodysenteriae ◽  
Oxford Nanopore ◽  
Sequencing Platforms ◽  
Sequence Types ◽  
Oxford Nanopore Technologies

The complete genomes of four Brachyspira hyodysenteriae isolates of the four different sequence types (STs) (ST6, ST66, ST196, and ST197) causing swine dysentery in Switzerland were generated by whole-genome sequencing and de novo hybrid assembly of reads obtained from second (Illumina) and third (Oxford Nanopore Technologies and Pacific Biosciences) high-throughput sequencing platforms.

scholarly journals NanoR: a user-friendly R package to analyze and compare nanopore sequencing data

2019 ◽  
Author(s):  
Davide Bolognini ◽  
Niccolò Bartalucci ◽  
Alessandra Mingrino ◽  
Alessandro Maria Vannucchi ◽  
Alberto Magi
Keyword(s):  
Real Time ◽  
Low Cost ◽  
R Package ◽  
Sequencing Data ◽  
High Performing ◽  
Dna And Rna ◽  
Oxford Nanopore ◽  
The One ◽  
User Friendly ◽  
Oxford Nanopore Technologies

AbstractMinION and GridION X5 from Oxford Nanopore Technologies are devices for real-time DNA and RNA sequencing. On the one hand, MinION is the only real-time, low cost and portable sequencing device and, thanks to its unique properties, is becoming more and more popular among biologists; on the other, GridION X5, mainly for its costs, is less widespread but highly suitable for researchers with large sequencing projects. Despite the fact that Oxford Nanopore Technologies’ devices have been increasingly used in the last few years, there is a lack of high-performing and user-friendly tools to handle the data outputted by both MinION and GridION X5 platforms. Here we present NanoR, a cross-platform R package designed with the purpose to simplify and improve nanopore data visualization. Indeed, NanoR is built on few functions but overcomes the capabilities of existing tools to extract meaningful informations from MinION sequencing data; in addition, as exclusive features, NanoR can deal with GridION X5 sequencing outputs and allows comparison of both MinION and GridION X5 sequencing data in one command. NanoR is released as free package for R at https://github.com/davidebolo1993/NanoR.

scholarly journals De novo genome assembly of the olive fruit fly (Bactrocera oleae) developed through a combination of linked-reads and long-read technologies

2018 ◽  
Author(s):  
Haig Djambazian ◽  
Anthony Bayega ◽  
Konstantina T. Tsoumani ◽  
Efthimia Sagri ◽  
Maria-Eleni Gregoriou ◽  
...  
Keyword(s):  
Y Chromosome ◽  
De Novo ◽  
Fruit Fly ◽  
Bactrocera Oleae ◽  
Olive Fruit Fly ◽  
Olive Fruit ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies

AbstractLong-read sequencing has greatly contributed to the generation of high quality assemblies, albeit at a high cost. It is also not always clear how to combine sequencing platforms. We sequenced the genome of the olive fruit fly (Bactrocera oleae), the most important pest in the olive fruits agribusiness industry, using Illumina short-reads, mate-pairs, 10x Genomics linked-reads, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT). The 10x linked-reads assembly gave the most contiguous assembly with an N50 of 2.16 Mb. Scaffolding the linked-reads assembly using long-reads from ONT gave a more contiguous assembly with scaffold N50 of 4.59 Mb. We also present the most extensive transcriptome datasets of the olive fly derived from different tissues and stages of development. Finally, we used the Chromosome Quotient method to identify Y-chromosome scaffolds and show that the long-reads based assembly generates very highly contiguous Y-chromosome assembly.JR is a member of the MinION Access Program (MAP) and has received free-of-charge flow cells and sequencing kits from Oxford Nanopore Technologies for other projects. JR has had no other financial support from ONT.AB has received re-imbursement for travel costs associated with attending Nanopore Community meeting 2018, a meeting organized my Oxford Nanopore Technologies.

scholarly journals Complete Closed Genome Sequence of the Inulin-Utilizing Lactiplantibacillus plantarum Strain Lp900, Obtained Using a Hybrid Nanopore and Illumina Assembly

2021 ◽  
Vol 10 (17) ◽  
Author(s):  
Jori Fuhren ◽  
Reindert Nijland ◽  
Michiel Wels ◽  
Jos Boekhorst ◽  
Michiel Kleerebezem
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Dna Sequence ◽  
Genome Sequence ◽  
De Novo ◽  
Circular Chromosome ◽  
Content Type ◽  
Diverse Species ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

Lactiplantibacillus plantarum is a genetically and phenotypically diverse species of lactic acid bacteria. We announce the hybrid de novo assembly of Oxford Nanopore Technologies and Illumina DNA sequence reads, producing a closed circular chromosome of 3,206,992 bp and six plasmids of the inulin-utilizing L. plantarum strain Lp900.

scholarly journals Fast and accurate de novo genome assembly from long uncorrected reads

2016 ◽  
Author(s):  
Robert Vaser ◽  
Ivan Sović ◽  
Niranjan Nagarajan ◽  
Mile Šikić
Keyword(s):  
Error Correction ◽  
De Novo ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Consensus Sequences ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Order Of Magnitude ◽  
Correction Step ◽  
Consensus Module

The assembly of long reads from Pacific Biosciences and Oxford Nanopore Technologies typically requires resource intensive error correction and consensus generation steps to obtain high quality assemblies. We show that the error correction step can be omitted and high quality consensus sequences can be generated efficiently with a SIMD accelerated, partial order alignment based stand-alone consensus module called Racon. Based on tests with PacBio and Oxford Nanopore datasets we show that Racon coupled with Miniasm enables consensus genomes with similar or better quality than state-of-the-art methods while being an order of magnitude faster.Racon is available open source under the MIT license at https://github.com/isovic/racon.git.

scholarly journals De novo clustering of long-read transcriptome data using a greedy, quality-value based algorithm

2018 ◽  
Author(s):  
Kristoffer Sahlin ◽  
Paul Medvedev
Keyword(s):  
Clustering Algorithm ◽  
De Novo ◽  
Substantial Improvement ◽  
Error Rates ◽  
Reconstruction Algorithms ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Transcript Reconstruction ◽  
Oxford Nanopore Technologies

AbstractLong-read sequencing of transcripts with PacBio Iso-Seq and Oxford Nanopore Technologies has proven to be central to the study of complex isoform landscapes in many organisms. However, current de novo transcript reconstruction algorithms from long-read data are limited, leaving the potential of these technologies unfulfilled. A common bottleneck is the dearth of scalable and accurate algorithms for clustering long reads according to their gene family of origin. To address this challenge, we develop isONclust, a clustering algorithm that is greedy (in order to scale) and makes use of quality values (in order to handle variable error rates). We test isONclust on three simulated and five biological datasets, across a breadth of organisms, technologies, and read depths. Our results demonstrate that isONclust is a substantial improvement over previous approaches, both in terms of overall accuracy and/or scalability to large datasets. Our tool is available at https://github.com/ksahlin/isONclust.

scholarly journals Performance of neural network basecalling tools for Oxford Nanopore sequencing

2019 ◽  
Author(s):  
Ryan R. Wick ◽  
Louise M. Judd ◽  
Kathryn E. Holt
Keyword(s):  
Neural Network ◽  
Electrical Signal ◽  
Specific Data ◽  
Consensus Sequences ◽  
Additional Signal ◽  
Oxford Nanopore ◽  
Majority Rules ◽  
Sequencing Platforms ◽  
Oxford Nanopore Technologies ◽  
Level Analysis

AbstractBasecalling, the computational process of translating raw electrical signal to nucleotide sequence, is of critical importance to the sequencing platforms produced by Oxford Nanopore Technologies (ONT). Here we examine the performance of different basecalling tools, looking at accuracy at the level of bases within individual reads and at majority-rules consensus basecalls in an assembly. We also investigate some additional aspects of basecalling: training using a taxon-specific dataset, using a larger neural network model and improving consensus basecalls in an assembly via additional signal-level analysis with Nanopolish. Training basecallers on taxon-specific data resulted in a significant boost in consensus accuracy, mostly due to the reduction of errors in methylation motifs. A larger neural network was able to improve both read and consensus accuracy, but at a cost to speed. Improving consensus sequences (‘polishing’) with Nanopolish somewhat negates the accuracy differences in basecallers, but pre-polish accuracy does have an effect on post-polish accuracy, so basecaller choice is still relevant even when Nanopolish is used.

scholarly journals De-novo Assembly of Limnospira fusiformis Using Ultra-Long Reads

2021 ◽  
Vol 12 ◽  
Author(s):  
McKenna Hicks ◽  
Thuy-Khanh Tran-Dao ◽  
Logan Mulroney ◽  
David L. Bernick
Keyword(s):  
Phylogenetic Analysis ◽  
Type Strain ◽  
Reference Genome ◽  
De Novo ◽  
Illumina Miseq ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies ◽  
Rdna Analysis

The Limnospira genus is a recently established clade that is economically important due to its worldwide use in biotechnology and agriculture. This genus includes organisms that were reclassified from Arthrospira, which are commercially marketed as “Spirulina.” Limnospira are photoautotrophic organisms that are widely used for research in nutrition, medicine, bioremediation, and biomanufacturing. Despite its widespread use, there is no closed genome for the Limnospira genus, and no reference genome for the type strain, Limnospira fusiformis. In this work, the L. fusiformis genome was sequenced using Oxford Nanopore Technologies MinION and assembled using only ultra-long reads (>35 kb). This assembly was polished with Illumina MiSeq reads sourced from an axenic L. fusiformis culture; axenicity was verified via microscopy and rDNA analysis. Ultra-long read sequencing resulted in a 6.42 Mb closed genome assembled as a single contig with no plasmid. Phylogenetic analysis placed L. fusiformis in the Limnospira clade; some Arthrospira were also placed in this clade, suggesting a misclassification of these strains. This work provides a fully closed and accurate reference genome for the economically important type strain, L. fusiformis. We also present a rapid axenicity method to isolate L. fusiformis. These contributions enable future biotechnological development of L. fusiformis by way of genetic engineering.

scholarly journals A fully phased accurate assembly of an individual human genome

2019 ◽  
Author(s):  
David Porubsky ◽  
Peter Ebert ◽  
Peter A. Audano ◽  
Mitchell R. Vollger ◽  
William T. Harvey ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Consensus Sequence ◽  
Error Rates ◽  
Single Individual ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Consensus Sequences ◽  
Oxford Nanopore ◽  
Genome Assemblies

The prevailing genome assembly paradigm is to produce consensus sequences that “collapse” parental haplotypes into a consensus sequence. Here, we leverage the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing (Strand-seq)1,2 and combine them with high-fidelity (HiFi) long sequencing reads3, in a novel reference-free workflow for diploid de novo genome assembly. Employing this strategy, we produce completely phased de novo genome assemblies separately for each haplotype of a single individual of Puerto Rican origin (HG00733) in the absence of parental data. The assemblies are accurate (QV > 40), highly contiguous (contig N50 > 25 Mbp) with low switch error rates (0.4%) providing fully phased single-nucleotide variants (SNVs), indels, and structural variants (SVs). A comparison of Oxford Nanopore and PacBio phased assemblies identifies 150 regions that are preferential sites of contig breaks irrespective of sequencing technology or phasing algorithms.

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