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Quantifying the biases in metagenome mining for realistic assessment of microbial ecology of naturally fermented foods

View Article: PubMed Central - PubMed

ABSTRACT

Cultivation-independent investigation of microbial ecology is biased by the DNA extraction methods used. We aimed to quantify those biases by comparative analysis of the metagenome mined from four diverse naturally fermented foods (bamboo shoot, milk, fish, soybean) using eight different DNA extraction methods with different cell lysis principles. Our findings revealed that the enzymatic lysis yielded higher eubacterial and yeast metagenomic DNA from the food matrices compared to the widely used chemical and mechanical lysis principles. Further analysis of the bacterial community structure by Illumina MiSeq amplicon sequencing revealed a high recovery of lactic acid bacteria by the enzymatic lysis in all food types. However, Bacillaceae, Acetobacteraceae, Clostridiaceae and Proteobacteria were more abundantly recovered when mechanical and chemical lysis principles were applied. The biases generated due to the differential recovery of operational taxonomic units (OTUs) by different DNA extraction methods including DNA and PCR amplicons mix from different methods have been quantitatively demonstrated here. The different methods shared only 29.9–52.0% of the total OTUs recovered. Although similar comparative research has been performed on other ecological niches, this is the first in-depth investigation of quantifying the biases in metagenome mining from naturally fermented foods.

No MeSH data available.


Comparison of alpha diversity differences between the two types of DNA mixing (DM, AM) and the specific individual methods in different fermented food types.(a) Venn diagram shows the difference in species-level OTU recovery between the mixes in comparison to the selected three individual methods for each food type. Values in the parentheses indicate extraction methods and numbers inside each region indicate the number of unique or shared OTUs. Variation among the extraction methods in the estimation of (b) Fisher’s alpha diversity and (c) Shannon’s diversity is represented as rarefaction plots. Replicates were used as mentioned elsewhere and the error bars represent standard error indicating the range of alpha diversity scores achieved at a given sampling depth.
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f5: Comparison of alpha diversity differences between the two types of DNA mixing (DM, AM) and the specific individual methods in different fermented food types.(a) Venn diagram shows the difference in species-level OTU recovery between the mixes in comparison to the selected three individual methods for each food type. Values in the parentheses indicate extraction methods and numbers inside each region indicate the number of unique or shared OTUs. Variation among the extraction methods in the estimation of (b) Fisher’s alpha diversity and (c) Shannon’s diversity is represented as rarefaction plots. Replicates were used as mentioned elsewhere and the error bars represent standard error indicating the range of alpha diversity scores achieved at a given sampling depth.

Mentions: Based on the above findings, we concluded that employing a single extraction method would lead to underestimation of the true microbial richness and diversity. A strategy was adapted to recover maximum microbial community and diversity by mixing the DNA of the individual methods in two different approaches. Equimolar mixing of metagenomic DNA (DM) and equimolar mixing of PCR amplicons (AM) generated from individual methods were subjected to MiSeq sequencing to investigate the impact of mixing on the recovery of the microbial community and diversity. Our results revealed that both types of mixing strategy (AM and DM) resulted in higher OTU recovery (53.1–68.2% in AM and 52.8–61.8% in DM of the total OTUs recovered by all the three methods together) (Fig. 5a). AM recovered higher (p < 0.05) species richness (Chao1) and Fisher’s alpha diversity than the other methods (Fig. 5, Supplementary Fig. S2 and Table S9) in all the food types except fermented soybean. Though Good’s coverage of 99.04 ± 0.6 in all cases indicated that our sequencing depth was sufficient to cover the high microbial diversity present in the fermented foods studied, the two mixing methods shared only 46.6–52.0% of the total OTUs recovered. Further, these mixing methods generated high number of additional OTUs (15–31.6% in AM and 9.4–20.1% in DM) which were not recovered by any of the individual methods (see Supplementary Table S10).


Quantifying the biases in metagenome mining for realistic assessment of microbial ecology of naturally fermented foods
Comparison of alpha diversity differences between the two types of DNA mixing (DM, AM) and the specific individual methods in different fermented food types.(a) Venn diagram shows the difference in species-level OTU recovery between the mixes in comparison to the selected three individual methods for each food type. Values in the parentheses indicate extraction methods and numbers inside each region indicate the number of unique or shared OTUs. Variation among the extraction methods in the estimation of (b) Fisher’s alpha diversity and (c) Shannon’s diversity is represented as rarefaction plots. Replicates were used as mentioned elsewhere and the error bars represent standard error indicating the range of alpha diversity scores achieved at a given sampling depth.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037447&req=5

f5: Comparison of alpha diversity differences between the two types of DNA mixing (DM, AM) and the specific individual methods in different fermented food types.(a) Venn diagram shows the difference in species-level OTU recovery between the mixes in comparison to the selected three individual methods for each food type. Values in the parentheses indicate extraction methods and numbers inside each region indicate the number of unique or shared OTUs. Variation among the extraction methods in the estimation of (b) Fisher’s alpha diversity and (c) Shannon’s diversity is represented as rarefaction plots. Replicates were used as mentioned elsewhere and the error bars represent standard error indicating the range of alpha diversity scores achieved at a given sampling depth.
Mentions: Based on the above findings, we concluded that employing a single extraction method would lead to underestimation of the true microbial richness and diversity. A strategy was adapted to recover maximum microbial community and diversity by mixing the DNA of the individual methods in two different approaches. Equimolar mixing of metagenomic DNA (DM) and equimolar mixing of PCR amplicons (AM) generated from individual methods were subjected to MiSeq sequencing to investigate the impact of mixing on the recovery of the microbial community and diversity. Our results revealed that both types of mixing strategy (AM and DM) resulted in higher OTU recovery (53.1–68.2% in AM and 52.8–61.8% in DM of the total OTUs recovered by all the three methods together) (Fig. 5a). AM recovered higher (p < 0.05) species richness (Chao1) and Fisher’s alpha diversity than the other methods (Fig. 5, Supplementary Fig. S2 and Table S9) in all the food types except fermented soybean. Though Good’s coverage of 99.04 ± 0.6 in all cases indicated that our sequencing depth was sufficient to cover the high microbial diversity present in the fermented foods studied, the two mixing methods shared only 46.6–52.0% of the total OTUs recovered. Further, these mixing methods generated high number of additional OTUs (15–31.6% in AM and 9.4–20.1% in DM) which were not recovered by any of the individual methods (see Supplementary Table S10).

View Article: PubMed Central - PubMed

ABSTRACT

Cultivation-independent investigation of microbial ecology is biased by the DNA extraction methods used. We aimed to quantify those biases by comparative analysis of the metagenome mined from four diverse naturally fermented foods (bamboo shoot, milk, fish, soybean) using eight different DNA extraction methods with different cell lysis principles. Our findings revealed that the enzymatic lysis yielded higher eubacterial and yeast metagenomic DNA from the food matrices compared to the widely used chemical and mechanical lysis principles. Further analysis of the bacterial community structure by Illumina MiSeq amplicon sequencing revealed a high recovery of lactic acid bacteria by the enzymatic lysis in all food types. However, Bacillaceae, Acetobacteraceae, Clostridiaceae and Proteobacteria were more abundantly recovered when mechanical and chemical lysis principles were applied. The biases generated due to the differential recovery of operational taxonomic units (OTUs) by different DNA extraction methods including DNA and PCR amplicons mix from different methods have been quantitatively demonstrated here. The different methods shared only 29.9&ndash;52.0% of the total OTUs recovered. Although similar comparative research has been performed on other ecological niches, this is the first in-depth investigation of quantifying the biases in metagenome mining from naturally fermented foods.

No MeSH data available.