scholarly journals High molecular weight DNA isolation method from diverse plant species for use with Oxford Nanopore sequencing

2019 ◽  
Author(s):  
Brieanne Vaillancourt ◽  
C. Robin Buell
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Isolation ◽  
Isolation Method ◽  
High Molecular Weight Dna ◽  
Sequencing Platform ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies

AbstractThe ability to generate long reads on the Oxford Nanopore Technologies sequencing platform is dependent on the isolation of high molecular weight DNA free of impurities. For some taxa, this is relatively straightforward; however, for plants, the presence of cell walls and a diverse set of specialized metabolites such as lignin, phenolics, alkaloids, terpenes, and flavonoids present significant challenges in the generation of DNA suitable for production of long reads. Success in generating long read lengths and genome assemblies of plants has been reported using diverse DNA isolation methods, some of which were tailored to the target species and/or required extensive labor. To avoid the need to optimize DNA isolation for each species, we developed a taxa-independent DNA isolation method that is relatively simple and efficient. This method expands on the Oxford Nanopore Technologies high molecular weight genomic DNA protocol from plant leaves and utilizes a conventional cetyl trimethylammonium bromide extraction followed by removal of impurities and short DNA fragments using commercially available kits that yielded robust N50 read lengths and yield on Oxford Nanopore Technologies flow cells.

scholarly journals High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0253830
Author(s):  
Ashley Jones ◽  
Cynthia Torkel ◽  
David Stanley ◽  
Jamila Nasim ◽  
Justin Borevitz ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Magnetic Bead ◽  
Read Length ◽  
High Molecular Weight Dna ◽  
Size Selection ◽  
Oxford Nanopore ◽  
Long Read ◽  
Selection For

Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30–50 kb, with reads exceeding 200 kb and outputs ranging from 15–30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria samples.

scholarly journals Long-read metagenomics retrieve complete single-contig bacterial genomes from canine feces

2021 ◽  
Author(s):  
Anna Cusco ◽  
Daniel Pérez ◽  
Joaquim Viñes ◽  
Norma Fàbregas ◽  
Olga Francino
Keyword(s):  
Molecular Weight ◽  
16S Rrna ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Bacterial Species ◽  
Ribosomal Genes ◽  
Bacterial Genomes ◽  
High Molecular Weight Dna ◽  
Long Reads ◽  
Long Read

Abstract BackgroundLong-read sequencing in metagenomics facilitates the assembly of complete genomes out of complex microbial communities. These genomes include essential biologic information such as the ribosomal genes or the mobile genetic elements, which are usually missed with short-reads. We applied long-read metagenomics with Nanopore sequencing to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a dog fecal sample.ResultsWe used nanopore long-read metagenomics and frameshift aware correction on a canine fecal sample and retrieved eight single-contig HQ MAGs, which were > 90% complete with < 5% contamination, and contained most ribosomal genes and tRNAs. At the technical level, we demonstrated that a high-molecular-weight DNA extraction improved the metagenomics assembly contiguity, the recovery of the rRNA operons, and the retrieval of longer and circular contigs that are potential HQ MAGs. These HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Catenibacterium, Blautia, and Enterococcus genera. Linking our results to previous gastrointestinal microbiome reports (metagenome or 16S rRNA-based), we found that some bacterial species on the gastrointestinal tract seem to be more canid-specific –Succinivibrio, Prevotellamassilia, Phascolarctobacterium, Blautia_A sp900541345–, whereas others are more broadly distributed among animal and human microbiomes –Sutterella, Catenibacterium, Enterococcus, and Blautia sp003287895. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Moreover, we show that long reads are essential to gain biological insights that are otherwise missed in short-read MAGs catalogs, as shown by the mobilome functions detected in the long-read HQ MAGs.ConclusionsWe recovered eight single-contig HQ MAGs from canine feces of a healthy dog with nanopore long-reads. We also retrieved relevant biological insights from these specific bacterial species previously missed in public databases, such as complete ribosomal operons and mobilome functions. The high-molecular-weight DNA extraction improved the assembly's contiguity, whereas the high-accuracy basecalling, the raw read error correction, the assembly polishing, and the frameshift correction reduced the insertions and deletion errors. Both experimental and analytical steps ensured the retrieval of complete bacterial genomes.

A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria)

1992 ◽  
Vol 38 (1) ◽  
pp. 65-68 ◽  
Author(s):  
Ken F. Jarrell ◽  
David Faguy ◽  
Anne M. Hebert ◽  
Martin L. Kalmokoff
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Thermal Denaturation ◽  
Dna Isolation ◽  
Methanogenic Archaea ◽  
Restriction Enzymes ◽  
Mole Percent ◽  
High Molecular Weight Dna ◽  
Isolation Methods ◽  
General Method

High molecular weight DNA was readily isolated from all methanogens treated, as well as from thermophilic anaerobic eubacteria, by grinding cells frozen in liquid N2, prior to lysis with SDS. DNA can subsequently be purified by the usual phenol–chloroform extractions. The procedure yields DNA readily cut by restriction enzymes and suitable for oligonucleotide probing, as well as for mole percent G + C content determination by thermal denaturation. The method routinely yields DNA of high molecular weight and is an improvement over DNA isolation methods for many methanogens, which often involve an initial breakage of the cells in a French pressure cell. Key words: methanogens, archaebacteria, archaea, DNA isolation.

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.

NUTRITIONAL AND THERAPEUTIC EVALUATION OF SPIRULINA PLATENSIS

2020 ◽  
pp. 86-90
Author(s):  
SACHIN KULKARNI ◽  
DEEPALI CHAVAN
Keyword(s):  
Molecular Weight ◽  
Protein Content ◽  
High Molecular Weight ◽  
Spirulina Platensis ◽  
Dna Isolation ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Growth Monitoring ◽  
High Molecular Weight Dna ◽  
Sds Page

Objective: The present investigation was to isolate and produce Spirulina platensis on high scale for food pharmaceutical and aquaculture due to the presence of high protein content. Methods: Cultivation of Spirulina preparation of culture medium, inoculums build up, growth monitoring, harvesting, drying, procedure for protein estimation, sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and DNA isolation of Spirulina platensis. Results: The study shows that protein content of S. platensis was found to be 62% protein profile was studied through SDS-PAGE, the bands were in the range of 35 kDa–44 kDa. S. platensis contain high molecular weight DNA. The bands was seen and visualize under the transilluminator it reveals that the alga S. platensis contain high molecular weight DNA. Conclusion: The production of Spirulina by simple pH determination method suggests economic production of alga by a simple process. The DNA isolation showing that these algae contain high molecular weight DNA. However, utilization of this biomass for varied end uses, such as food, feed, aquaculture, and pharmaceuticals.

scholarly journals Scalable high-molecular weight DNA extraction for long-read sequencing v1 (protocols.io.bnjhmcj6)

protocols.io ◽  
2020 ◽  
Author(s):  
Ashley Jones ◽  
Cynthia Torkel ◽  
David Stanley ◽  
Jamila Nasim ◽  
Justin Borevitz ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
High Molecular Weight Dna ◽  
Long Read

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.

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