Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Synthesis of Selenium Nanoparticles by Bacillus laterosporus Using Gamma Radiation.

Aim: This study shows the possible synthesis of Selenium Nanoparticles (SeNPs) in aerobic optimized conditions using Bacillus laterosporus (B. laterosporus) bacterial strain. Methodology: B. laterosporus was used to reduce selenium ions (selenite anions) to SeNPs by fermentation in Luria-Bertani Enrichment (EM) medium. Optimization of fermentation conditions using two-level full factorial design was performed. SeNPs were further characterized by UV-Vis., DLS, TEM, FT-IR, EDX and XRD analysis. SeNPs synthesis by Gamma irradiated B. laterosporus cells at different radiation doses was reported. Evaluation the probability of B. laterosporus to synthesis SeNPs by fermentation in skimmed milk aerobically. A microtiterplate assay was used to evaluate the ability of SeNPs to inhibit the biofilm formation of Pseudomonas aeruginosa. Evaluating the antimicrobial activity of some antibiotic agents upon addition of SeNPs was performed. Results: B. laterosporus reduced the soluble, toxic, colorless selenium ions to the insoluble, non-toxic, red elemental SeNPs. Statistical analysis showed that the results were normally distributed. Temperature, incubation period and pH were significant factors in the fermentation process, in which the maximum SeNPs produced (8.37μmole/ml) was at temperature 37ºC, incubation period 48hr, pH7. The Gamma radiation exposure dose 1.5kGy gave the maximum SeNPs produced (10.01 μmole/ml). A pink color appear in the fermented milk revealing the formation of SeNPs-enriched milk. SeNPs inhibit the biofilm formation of Pseudomonas aeruginosa with a percentage reduction of 99.7%. SeNPs increase the antibacterial activity of fucidic acid by 13.6% and 28.5% against Escherichia coli and Staphylococcus aureus respectively. But with Gentamycin sulphate, no change in the antibacterial activity. Conclusion: SeNPs can be synthesized aerobically by the probiotic B. laterosporus bacterial strain. SeNPs can be incorporated in nutraceuticals and functional foods like milk also can be used to inhibit the bacterial biofilm formation and can be added to some antibacterial creams to enhance their antimicrobial activity.