scholarly journals HMW DNA extraction from diverse plants species for PacBio and Nanopore sequencing v1 (protocols.io.5t7g6rn)

protocols.io ◽  
2019 ◽  
Author(s):  
Alessia Russo ◽  
Giacomo Potente ◽  
Baptiste Mayjonade
Keyword(s):  
Dna Extraction ◽  
Nanopore Sequencing ◽  
Hmw Dna

scholarly journals Optimizing DNA extraction methods for Nanopore sequencing of Neisseria gonorrhoeae direct from urine samples

2019 ◽  
Author(s):  
Teresa L. Street ◽  
Leanne Barker ◽  
Nicholas D. Sanderson ◽  
James Kavanagh ◽  
Sarah Hoosdally ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Reference Genome ◽  
Extraction Methods ◽  
Urine Samples ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Human Dna ◽  
Whole Genomes ◽  
Dna Extraction Methods ◽  
Multiple Antibiotics

AbstractBackgroundEmpirical gonorrhoea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance.MethodsWe investigated if Nanopore sequencing can detect sufficient N. gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae spiked urine samples and samples from gonorrhoea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced whilst minimizing contaminating host DNA.ResultsIn simulated infections the Qiagen UCP Pathogen Mini kit provided the highest ratio N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections, but decreased yields in clinical samples. In ten urine samples from men with symptomatic urethral gonorrhoea, ≥87% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥92% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR Media tubes and from urethral swabs, and in the presence of simulated Chlamydia co-infection.ConclusionUsing Nanopore sequencing of urine samples from men with urethral gonorrhoea sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

scholarly journals From low cost plant HMW DNA extraction to MinION sequencing v1

protocols.io ◽  
2021 ◽  
Author(s):  
Julien Serret ◽  
marie.couderc not provided ◽  
Cedric Mariac ◽  
Laurencealbar not provided ◽  
Francois Sabot
Keyword(s):  
Dna Extraction ◽  
Low Cost ◽  
Hmw Dna

scholarly journals Exploring Quantitative Metagenomics Studies using Oxford Nanopore Sequencing: A Computational and Experimental Protocol

2020 ◽  
Author(s):  
Rohia ALILI ◽  
Eugeni BELDA ◽  
Karine CLEMENT ◽  
Phuong Le ◽  
Edi PRIFTI ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Gut Microbiome ◽  
Large Scale ◽  
Low Cost ◽  
Read Length ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Experimental Protocol ◽  
Microbial Composition ◽  
Oxford Nanopore

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.

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

protocols.io ◽  
2019 ◽  
Author(s):  
Natalie Solonenko ◽  
Marie Burris
Keyword(s):  
Dna Extraction ◽  
Hmw Dna

FindingNemo Extraction 2: Phenol-free Method v2

2021 ◽  
Author(s):  
Inswasti Cahyani ◽  
John Tyson ◽  
Nadine Holmes ◽  
Josh Quick ◽  
Nicholas Loman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Dna Extraction ◽  
Human Cell ◽  
Extraction Method ◽  
Human Cell Line ◽  
Free Phenol ◽  
Hmw Dna ◽  
Lysis Buffer ◽  
Dna Precipitation ◽  
Ultra High Molecular Weight

This is a sub-protocol designed to extract/isolate ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers using a phenol-free extraction method. A DNA extraction protocol that yields clean and homogeneous UHMW DNA is important for a good UL sequencing output. The choice of protocol should be based on achieving these parameters. Kit-free, phenol-free protocol is a modification of NEB's Monarch HMW DNA Extraction Kit for Cells & Blood, with the option to use SDS or CTAB in the lysis buffer. This protocol also uses glass beads for DNA precipitation matrix. We tested this sub-protocol in human cell line, with input cells of 3 millions but can be varied from 1-5 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.

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