Isolation and Cloning of High-Molecular-Weight Metagenomic DNA from Soil Microorganisms

Liles, Mark R.; Williamson, Lillie D.; Rodbumrer, Jitsupang; Torsvik, Vigdis; Parsley, Larissa C.; Goodman, Robert M.; Handelsman, Jo

doi:10.1101/pdb.prot5271

Cited by 14 publications

(10 citation statements)

References 15 publications

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“…The samples were immediately transported to laboratory and were passed through 0.22 μm Sterivex filters (Millipore) using a peristaltic pump for concentrating the biomass. The filters were stored overnight at −20°C temperature and environmental DNA was extracted the very next day using standard published protocols (Tringe and Rubin, 2005; Liles et al, 2009). After extraction, the DNA was quantified on an UV-Vis spectrophotometer (Beckman DU730).…”

Section: Methodsmentioning

confidence: 99%

Diversity of arsenite oxidizing bacterial communities in arsenic-rich deltaic aquifers in West Bengal, India

Ghosh¹,

Bhadury²,

Routh³

2014

Front. Microbiol.

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High arsenic (As) concentration in groundwater has affected human health, particularly in South-East Asia putting millions of people at risk. Biogeochemical cycling of As carried out by different bacterial groups are suggested to control the As fluxes in aquifers. A functional diversity approach in link with As precipitation was adopted to study bacterial community structures and their variation within the As contaminated Bengal Delta Plain (BDP) aquifers of India. Groundwater samples collected from two shallow aquifers in Karimpur II (West Bengal, India), during years 2010 and 2011, were investigated to trace the effects immediately after monsoon period (precipitation) on community structure and diversity of bacterial assemblages with a focus on arsenite oxidizing bacterial phyla for two successive years. The study focused on amplification, clone library generation and sequencing of the arsenite oxidase large sub-unit gene aioA and 16S rRNA marker, with respect to changes in elemental concentrations. New set of primers were designed to amplify the aioA gene as a phylogenetic marker to study taxonomically diverse arsenite oxidizing bacterial groups in these aquifers. The overall narrow distribution of bacterial communities based on aioA and 16S rRNA sequences observed was due to poor nutrient status and anoxic conditions in these As contaminated aquifers. Proteobacteria was the dominant phylum detected, within which Acidovorax, Hydrogenophaga, Albidiferax, Bosea, and Polymorphum were the major arsenite oxidizing bacterial genera based on the number of clones sequenced. The structure of bacterial assemblages including those of arsenite oxidizing bacteria seems to have been affected by increase in major elemental concentrations (e.g., As, Fe, S, and Si) within two sampling sessions, which was supported by statistical analyses. One of the significant findings of this study is detection of novel lineages of 16S rRNA-like bacterial sequences indicating presence of indigenous bacterial communities BDP wells that can play important role in biogeochemical cycling of elements including As.

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Section: Methodsmentioning

confidence: 99%

Diversity of arsenite oxidizing bacterial communities in arsenic-rich deltaic aquifers in West Bengal, India

Ghosh¹,

Bhadury²,

Routh³

2014

Front. Microbiol.

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“…Manual metagenomic DNA extraction protocols developed by Zhou et al 1996;Wechter et al 2003;Brady 2007;Amorim et al 2008;Pang et al 2008;Liles et al 2009;Inceoglu et al 2010 were applied for environmental DNA extraction. DNA extracted from multiple methods (n = 3) was pooled and diluted (1/100 dilutions) with final concentration of 20 ng/µl and used as template for PCR amplification.…”

Section: Metagenomic Dna Extraction and Amplificationmentioning

confidence: 99%

Bacterial diversity of Drass, cold desert in Western Himalaya, and its comparison with Antarctic and Arctic

et al. 2015

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Drass is the coldest inhabited place in India and the second coldest, inhabited place in the world, after Siberia. Using the 16SrDNA amplicon pyrosequencing, bacterial diversity patterns were cataloged across the Drass cold desert. In order to identify the ecotype abundance across cold desert environment, bacterial diversity patterns of Drass were further compared with the bacterial diversity of two other cold deserts, i.e., Antarctic and Arctic. Acidobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and Gemmatimonadetes were among the highly abundant taxonomic groups present across all the three cold deserts and were designated as the core phyla. However, Firmicutes, Nitrospirae, Armatimonadetes (former candidate division OP10), Planctomycetes, TM7, Chloroflexi, Deinococcus-Thermus, Tenericutes and candidate phyla WS3 were identified as rare phyla in Drass, Antarctic and Arctic samples. Differential abundance patterns were also computed across all the three samples, i.e., Acidobacteria (32.1 %) were dominant in Drass and Firmicutes (52.9 ± 17.6 %) and Proteobacteria (42 ± 1.3 %) were dominant in Antarctic and Arctic reference samples, respectively. Alpha diversity values Shannon's (H) and Simpson's (1-D) diversity indices were highest for Antarctic samples, whereas richness estimators (ACE and Chao1) were maximum for Drass soil suggesting greater species richness in bacterial communities in Drass than the Antarctic and Arctic samples.

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“…One common requirement for the different metagenomic approaches is DNA. It is important to isolate high purity (OD 260 /OD 280 absorbance ratio of 1.8–2) and high molecular weight (HMW) DNA for successful library construction . Obtaining HMW DNA allows for more opportunities to find genes of interest .…”

Section: Introductionmentioning

confidence: 99%

“…8 Obtaining HMW DNA allows for more opportunities to find genes of interest. 8 However, it is difficult to isolate HMW bacterial DNA with high purity through direct DNA extraction techniques as DNA extracted by in situ lysis could be associated with organic matter 9,10 limiting its recovery. Coisolation of contaminants, eukaryotic, and extracellular DNA poses major obstacles during the construction of prokaryotic metagenomic libraries from environmental samples.…”

Section: Introductionmentioning

confidence: 99%

Selective isolation of bacteria for metagenomic analysis: Impact of membrane characteristics on bacterial filterability

Nnadozie

Lin

Govinden

2015

Biotechnology Progress

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For indirect DNA extraction for metagenomics studies, bacterial cells can be effectively separated from sample debris by using a simple size exclusion technique, such as filtration, and thereafter lysed. The requirement for the optimal recovery of cells in filtrates is critical to achieve sufficient DNA yield and a representative population. Particles smaller than the filter pore size are expected to be found in the filtrate, whereas particles larger than the filter pore sizes are excluded. However, this is not always the case. It is established that the membrane pore size influences filtration efficiency to some degree. In addition the physicochemical characteristics of the filter suspension and characteristics of the microbial cells being filtered influence the exclusion property of a membrane. This review provides an overview of membrane filtration techniques and the factors that affect filterability of bacteria cells through a filter membrane.

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Isolation and Cloning of High-Molecular-Weight Metagenomic DNA from Soil Microorganisms

Cited by 14 publications

References 15 publications

Diversity of arsenite oxidizing bacterial communities in arsenic-rich deltaic aquifers in West Bengal, India

Diversity of arsenite oxidizing bacterial communities in arsenic-rich deltaic aquifers in West Bengal, India

Bacterial diversity of Drass, cold desert in Western Himalaya, and its comparison with Antarctic and Arctic

Selective isolation of bacteria for metagenomic analysis: Impact of membrane characteristics on bacterial filterability

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