From low cost plant HMW DNA extraction to MinION sequencing v1

Abstract Background: The gut microbiome plays a major role in chronic diseases, several of which are characterized by an altered diversity and composition of bacterial communities. Large-scale sequencing projects allowed the characterization of these microbial community perturbations. However, a gap remains in how these discoveries can be translated into clinical applications. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed.Results: Here, we propose a computational and experimental protocol for whole genome quantitative metagenomics studies of the human gut microbiome with Oxford Nanopore sequencing technology (ONT). We developed a bioinformatic pipeline to process ONT sequences based on the evaluation of different alignment parameters in the estimation of microbial diversity and composition. We also optimized stool collection and DNA extraction methods to maximize read length, a critical parameter for the sequence alignment and classification. Our analytical pipeline was evaluated using simulations of metagenomic communities to reflect naturally occuring compositional variations. We then validated our experimental and analytical pipeline with stool samples from a bariatric surgery cohort sequenced with ONT and Illumina, revealing comparable diversity and microbial composition profiles. These results were compared to those previously obtained with SOLiD sequencing, where differences were observed, possibly explained by variations in library preparation steps. Finally, we found that sequences obtained with ONT allowed assembly of complete genomes for disease-related species.Conclusion: This protocol can be implemented in the clinical or individual setting, bringing rapid personalized whole genome profiling of target microbiome species. Keywords: quantitative metagenomics, microbiome, obesity, gut microbiota, microbial DNA extraction, sequencing, Simulation, Oxford Nanopore Technologies, MinION.

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Comparison of the efficiency of some of the most usual DNA extraction methods for woody plants in different tissues of Vitis vinifera L.

OENO One ◽

10.20870/oeno-one.2013.47.4.1559 ◽

2013 ◽

Vol 47 (4) ◽

pp. 227 ◽

Cited By ~ 1

Author(s):

Gemma Marsal ◽

Núria Boronat ◽

Joan Miquel Canals ◽

Fernando Zamora ◽

Francesca Fort

Keyword(s):

Vitis Vinifera ◽

Dna Extraction ◽

Woody Plants ◽

Extraction Method ◽

Low Cost ◽

Handling Time ◽

Extraction Methods ◽

Original Method ◽

Dna Extraction Method ◽

Functional Dna

Aim: To compare different methods for extracting DNA from non-recalcitrant and recalcitrant tissues of Vitis vinifera woody plants and propose a modification of a previously published method to reduce the time and cost of extraction.Methods and results: DNA was extracted from young and mature leaves as well as from stems and seeds using some of the most common methods of DNA isolation and two commercial kits. Another commercial kit, which does not require DNA extraction prior to PCR, was also used. Only two methods provided adequate results in all tissues. Other methods were only applicable to some tissues and some did not yield any functional DNA in any tissue. A modification of the method reported by Marsal et al. (2011) is proposed to reduce handling time and cost.Conclusion: All of the methods studied here use a surfactant to improve the extractions. For DNA extraction from recalcitrant tissues to be optimal, it is best to use a combination of dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB). The changes made to the protocol reported by Marsal et al. (2011) enable functional DNA to be obtained from leaves in only 90 minutes and at very low cost (17 €/8 samples). However, this method cannot adequately isolate DNA from recalcitrant tissues (stems and seeds) and so, for this type of sample, we would recommend using the original method.Significance and impact of the study: Nowadays, handling time and cost are key factors in selecting the most suitable DNA extraction method. This study compares not only the effectiveness of the various methods but also the handling time and cost. It also proposes a modification of the fastest and most economic DNA extraction method for leaves so that handling time and processing cost will be reduced even further.

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Comparing the efficiency of six common methods for DNA extraction from root-lesion nematodes (Pratylenchus spp.)

Nematology ◽

10.1163/15685411-bja10049 ◽

2020 ◽

pp. 1-9

Author(s):

Valeria Orlando ◽

Simon G. Edwards ◽

Roy Neilson ◽

Tom Prior ◽

David Roberts ◽

...

Keyword(s):

Dna Extraction ◽

Crop Yields ◽

Low Cost ◽

Dna Amplification ◽

Extraction Methods ◽

Parasitic Nematodes ◽

Potential Threat ◽

Accurate Identification ◽

Lesion Nematodes ◽

Root Lesion Nematodes

Summary Robust and accurate identification of root-lesion nematodes (Pratylenchus spp.) is an essential step for determining their potential threat to crop yields and, consequently, development of an efficient agronomic management strategy. It is recognised that DNA-based techniques provide rapid identification of a range of plant-parasitic nematodes including Pratylenchus spp. Efficient and repeatable DNA extraction is central to molecular methodologies. Here, six common DNA extraction protocols were compared to evaluate their efficiency to obtain quality DNA samples for Pratylenchus penetrans. Samples with five and ten individuals of P. penetrans were successfully extracted and amplified by all extraction methods tested, whereas samples with a single nematode presented challenges for DNA amplification. Among all methods tested, the DNA extraction protocol with glass beads proved to be efficient for P. penetrans and all other species tested (P. crenatus, P. neglectus and P. thornei), generating high quality DNA at comparatively low cost and with a rapid sample throughput.

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Versatile hybrid acoustic micromixer with demonstration of circulating cell-free DNA extraction from sub-ml plasma samples

Lab on a Chip ◽

10.1039/c9lc01130g ◽

2020 ◽

Vol 20 (4) ◽

pp. 741-748 ◽

Cited By ~ 4

Author(s):

Alvaro J. Conde ◽

Ieva Keraite ◽

Alfredo E. Ongaro ◽

Maïwenn Kersaudy-Kerhoas

Keyword(s):

Dna Extraction ◽

Low Cost ◽

Plasma Samples ◽

Cell Free Dna ◽

Free Dna ◽

Circulating Cell Free Dna

A low-cost and easy to implement acoustic micromixer compatible with multiple fabrication technologies that can provide efficient and vigorous mixing.

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Whole genome sequencing of marine organisms by Oxford Nanopore Technologies: Assessment and optimization of HMW‐DNA extraction protocols

Ecology and Evolution ◽

10.1002/ece3.8447 ◽

2021 ◽

Author(s):

Sonia Boughattas ◽

Dana Albatesh ◽

Albandari Al‐Khater ◽

Bruno W. Giraldes ◽

Asma A. Althani ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Dna Extraction ◽

Genome Sequencing ◽

Marine Organisms ◽

Whole Genome ◽

Oxford Nanopore ◽

Hmw Dna ◽

Oxford Nanopore Technologies

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Extracting the invisible: obtaining high quality DNA is a challenging task in small arthropods

PeerJ ◽

10.7717/peerj.6753 ◽

2019 ◽

Vol 7 ◽

pp. e6753 ◽

Cited By ~ 9

Author(s):

Andrea Lienhard ◽

Sylvia Schäffer

Keyword(s):

Dna Extraction ◽

Low Cost ◽

Extraction Methods ◽

Chelating Resin ◽

Precipitation Method ◽

Ammonium Bromide ◽

User Friendliness ◽

Silica Membrane ◽

High Quality ◽

Dna Extraction Methods

BackgroundThe application of an appropriate extraction method is a relevant factor for the success of all molecular studies.MethodsSeven different DNA extraction methods suitable for high-throughput DNA sequencing with very small arthropods were compared by applying nine different protocols: three silica gel based spin methods, two cetyltrimethyl ammonium bromide (CTAB) based ones (one with an additional silica membrane), a protein precipitation method and a method based on a chelating resin (applying different protocols). The quantity (concentration) and quality (degradation, contamination, polymerase chain reaction (PCR) and sequencing success) of the extracted DNA as well as the costs, preparation times, user friendliness, and required supplies were compared across these methods. To assess the DNA quantity, two different DNA concentration measurements were applied. Additionally, the effect of varying amounts of starting material (different body sizes), variable lysis temperatures and mixing during DNA extraction was evaluated.ResultsAlthough low DNA concentrations were measured for all methods, the results showed that—with the exception of two methods—the PCR success was 100%. However, other parameters show vast differences. The time taken to perform DNA extraction varied from 20 min to 2.5 h (Chelex vs. CTAB) and the costs from 0.02 to 3.46 € (Chelex vs. QIAamp kit) per sample. High quality genomic DNA was only gained from four methods. Results of DNA quantity measurements further indicated that some devices cannot deal with small amounts of DNA and show variant results.DiscussionIn conclusion, using Chelex (chelating resin) turned out as a rapid, low-cost method which can provide high quality DNA for different kinds of molecular investigations.

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DNA extraction for HMW DNA v1 (protocols.io.6cbhasn)

protocols.io ◽

10.17504/protocols.io.6cbhasn ◽

2019 ◽

Author(s):

Natalie Solonenko ◽

Marie Burris

Keyword(s):

Dna Extraction ◽

Hmw Dna

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A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

AIMS Microbiology ◽

10.3934/microbiol.2021019 ◽

2021 ◽

Vol 7 (3) ◽

pp. 304-319

Author(s):

Spyridon Andreas Papatheodorou ◽

◽

Panagiotis Halvatsiotis ◽

Dimitra Houhoula ◽

Keyword(s):

Dna Extraction ◽

Foodborne Pathogens ◽

Extraction Method ◽

Pathogenic Bacteria ◽

Low Cost ◽

Isoamyl Alcohol ◽

Extraction Methods ◽

Molecular Techniques ◽

Bacterial Dna ◽

Dna Extraction Method

<abstract> Foodborne infections continue to plague Europe. Food safety monitoring is in crisis as the existing techniques for detecting pathogens do not keep up with the global rising of food production and consumption. Thus, the development of innovative techniques for detecting and identifying pathogenic bacteria has become critical. The aim of the present study was firstly to develop an innovative simple and low cost method of extracting bacterial DNA from contaminated food and water samples with <italic>Salmonella enteric</italic> subsp. <italic>enteric</italic> serovar Typhimurium and <italic>Listeria monocytogenes</italic> and its comparison with two commercial DNA extraction kits (Qiagen, Macherey-Nagel). Finally, pathogens' detection using two molecular techniques (PCR-electrophoresis, LAMP), in order to evaluate the best combination of DNA extraction and identification based on their sensitivity, cost, rapidity and simplicity. Considering the above criteria, among them, best was proved an in-house bacterial DNA extraction method, based on the chloroform-isoamyl alcohol protocol, with certain modifications. This technique showed statistically similar results in terms of sensitivity, compared to the commercial kits, while at the same time maintained high rapidity and much lower cost. Lastly, between the molecular techniques, LAMP was found more promising considering its simplicity, high rapidity and sensitivity. Conclusively, the in-house DNA extraction method along with the LAMP technique, was proven to be the best among the presented combinations. </abstract>

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