High Molecular Weight genomic DNA extraction from Gram-negative bacteria for long reads sequencing (Xanthomonas ssp.) v1 (protocols.io.r9id94e)

protocols.io ◽  
2018 ◽  
Author(s):  
Baptiste Mayjonade
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Genomic Dna Extraction ◽  
Long Reads

scholarly journals A simple method for high molecular-weight genomic DNA extraction suitable for long-read sequencing from spores of an obligate biotroph oomycete

2020 ◽  
Vol 178 ◽  
pp. 106054
Author(s):  
Charlotte Penouilh-Suzette ◽  
Sandra Fourré ◽  
Guillaume Besnard ◽  
Laurence Godiard ◽  
Yann Pecrix
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Simple Method ◽  
Genomic Dna Extraction ◽  
Long Read ◽  
Obligate Biotroph

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.

scholarly journals Structure and function of a novel osmoregulated periplasmic fiber-forming high-molecular-weight carbohydrate of Myxococcus xanthus

2020 ◽  
Author(s):  
Sapeckshita Agrawal ◽  
Christian Heiss ◽  
David M. Zuckerman ◽  
Jeffery M. T. So ◽  
Koen Semeijn ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cell Envelope ◽  
Myxococcus Xanthus ◽  
Soil Bacterium ◽  
Model Organisms ◽  
Low Molecular Weight ◽  
Gram Negative Bacteria ◽  
Vegetative Cells ◽  
Gram Negative

AbstractOsmoregulation is of central importance for living cells. In Gram-negative bacteria, strategies for osmoregulation and turgor maintenance in hypotonic environments include the synthesis, accumulation, and modification of periplasmic oligosaccharides. These osmoregulated periplasmic glucans (OPGs, formerly known as membrane-derived oligosaccharides or MDOs) promote water uptake and retention, keeping the cells in an optimal state of hydration. While our understanding of OPG-dependent osmoregulation in a number of model organisms like Escherichia coli is quite detailed, less is known about these processes in bacteria that live in environments characterized by strongly fluctuating osmolarity, such as soil. Here we describe that the soil bacterium Myxococcus xanthus lacks a canonical low-molecular-weight OPG, but instead possesses a novel high-molecular-weight, fiber-forming polysaccharide. Chemical analysis reveals that this polysaccharide is several thousand kilodaltons in size, composed of a highly branched decasaccharide repeat unit containing mannose, glucose, N-acetylglucosamine, and rhamnose. Physiological experiments indicate that the polysaccharide is osmoregulated thereby functionally replacing the canonical OPG. Moreover, experiments indicate that this high-molecular-weight periplasmic polysaccharide forms a fibrillar meshwork that stabilizes the cell envelope during glycerol spore formation, a process during which the entire peptidoglycan of the cell is degraded and the rod-shaped vegetative cells convert into spherical spores.SignificanceOsmoprotection is a necessity for every living cell, particularly in an environment with fluctuating osmolarity. In Gram-negative bacteria, low-molecular-weight osmoregulated periplasmic glucans (OPGs) are an important component of the osmotic stress response in hypotonic environments. Here, we describe that the soil bacterium Myxococcus xanthus does not possess such an OPG but instead accumulates a novel high-molecular-weight fiber-forming polysaccharide in the periplasm in response to hypotonic conditions. This polymer is important for osmoprotection of the cells and plays a key role in the stabilization of the cell envelope during the conversion of rod-shaped vegetative cells into spherical spores. These results indicate that bacteria may use non-OPG carbohydrates for osmoprotection and cell wall stabilization during processes like cellular differentiation.

scholarly journals Genomic DNA isolation from scrophularieae dried leaves using a simple, high-throughput protocol

2019 ◽  
Vol 48 (4) ◽  
pp. 1231-1235
Author(s):  
Mehrshid Riahi ◽  
Melina Babaei ◽  
Farrokh Ghahremaninejad
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
High Throughput ◽  
Genomic Dna ◽  
Dna Isolation ◽  
High Molecular Weight Dna ◽  
Plant Dna ◽  
Genomic Dna Isolation

This communication described efficient DNA extraction from Scrophularia and Verbascum samples. Modified Murray and Thompson modified Cota-Sànchez method and Bioflux kit methods were applied for the extraction of DNA. Among the different methods, Bioflux kit Plant DNA extraction kit, coupled with some modification was the best for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods.

scholarly journals GENOMIC DNA EXTRACTION METHODS: A COMPARATIVE CASE STUDY WITH GRAM-NEGATIVE ORGANISMS

2015 ◽  
Vol VI (11) ◽  
pp. 61-68 ◽  
Author(s):  
Sanjida JAHAN JAHAN ◽  
Sayeeda Fahmee CHOWDHURY ◽  
Shahida Akter MITU ◽  
Mohammad SHAHRIA SHAHRIAR ◽  
Mohiuddin Ahmed BH BHUIYAN
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Methods ◽  
Comparative Case Study ◽  
Gram Negative ◽  
Genomic Dna Extraction ◽  
Gram Negative Organisms ◽  
Dna Extraction Methods

Optimization of genomic DNA extraction from formalin-fixed fish specimens

2013 ◽  
Vol 19 (6) ◽  
pp. 1068-1073
Author(s):  
Xiaolan KONG ◽  
Zuozhi CHEN ◽  
Lin LIN ◽  
Chunhou LI ◽  
Peiwen LIANG
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Genomic Dna Extraction ◽  
Formalin Fixed

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool
Keyword(s):  
Dna Extraction ◽  
Control Method ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Yield ◽  
Gram Negative Bacteria ◽  
Bacterial Dna ◽  
Gram Negative ◽  
E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

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