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.

Comparison of DNA Extraction Methods for Human Oral Microbiome Research.

The oral micro biome is highly diverse and its composition is associated with oral disease and potentially diseases at other sites. Our objective is to evaluate DNA extraction methods potentially suitable for population-based investigations on the oral human microbiome and disease risk. Six commonly used microbial DNA extraction kits, employing either enzymatic methods or mechanical bead beating for cell lysis, were evaluated for the following aspects total DNA yield and quality and 16s rRNA DNA product and representation of microbial diversity. All analyses were carried out using a pooled and homogenized sample from one study subject. 16s rRNA gene sequence data were processed using the QIIME pipeline. One way ANOVA and Kruskal-Wallis tests were used to compare the different DNA extraction methods. We found that enzymatic extraction kits produced higher human genomic DNA, compared with mechanical extraction kits, however, phylogenic diversity in oral microbiome community structure from 16s rRNA gene sequence reads revealed no important differences between kit types. Enzymatic and mechanical bead beating kits provide alternative approaches for DNA extraction of oral microbiome DNA from oral wash samples. Greater total DNA yields are found in enzymatic approaches but microbial diversity can be similarly well characterized by either enzymatic or mechanical bead beating approaches.