Limits...
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.


Variation in the metagenomic DNA yield of different extraction methods (I–VIII) as observed in fermented (a) soybean (b) milk (c) fish and (d) bamboo shoot. Each method represents data from ten independent replicates. ANOVA highlights the overall significant difference in the DNA yield between extraction methods for each food type. The p value for pairwise comparison of the extraction methods in DNA yield is listed in Supplementary Table S1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037447&req=5

f1: Variation in the metagenomic DNA yield of different extraction methods (I–VIII) as observed in fermented (a) soybean (b) milk (c) fish and (d) bamboo shoot. Each method represents data from ten independent replicates. ANOVA highlights the overall significant difference in the DNA yield between extraction methods for each food type. The p value for pairwise comparison of the extraction methods in DNA yield is listed in Supplementary Table S1.

Mentions: The differences in the metagenomic DNA recovery from varied types of fermented foods by eight different extraction methods are shown in Fig. 1. Higher DNA recovery in fermented soybean (4.5–47.8 μg.g−1) and milk products (5.28–24.49 μg.g−1) compared to fermented bamboo shoot (1.83–9.28 μg.g−1) and fish products (0.5–6.18 μg.g−1) was observed. In general, the enzymatic methods (II, V) recovered maximum DNA yield from fermented soybean and milk products whereas the non-enzymatic method-I recovered maximum DNA from the fermented bamboo shoot and fish products. Across the food types, the in-house developed methods (III, IV) and the commercial method (VI) showed poor DNA recovery. The DNA recovered by all the methods were of good quality (A260/280 = 1.7–2.3) with no PCR inhibitors. For each food type, the efficiency of DNA recovery strongly depended on the extraction method applied and a single method cannot efficiently extract DNA from all the food types.


Quantifying the biases in metagenome mining for realistic assessment of microbial ecology of naturally fermented foods
Variation in the metagenomic DNA yield of different extraction methods (I–VIII) as observed in fermented (a) soybean (b) milk (c) fish and (d) bamboo shoot. Each method represents data from ten independent replicates. ANOVA highlights the overall significant difference in the DNA yield between extraction methods for each food type. The p value for pairwise comparison of the extraction methods in DNA yield is listed in Supplementary Table S1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Variation in the metagenomic DNA yield of different extraction methods (I–VIII) as observed in fermented (a) soybean (b) milk (c) fish and (d) bamboo shoot. Each method represents data from ten independent replicates. ANOVA highlights the overall significant difference in the DNA yield between extraction methods for each food type. The p value for pairwise comparison of the extraction methods in DNA yield is listed in Supplementary Table S1.
Mentions: The differences in the metagenomic DNA recovery from varied types of fermented foods by eight different extraction methods are shown in Fig. 1. Higher DNA recovery in fermented soybean (4.5–47.8 μg.g−1) and milk products (5.28–24.49 μg.g−1) compared to fermented bamboo shoot (1.83–9.28 μg.g−1) and fish products (0.5–6.18 μg.g−1) was observed. In general, the enzymatic methods (II, V) recovered maximum DNA yield from fermented soybean and milk products whereas the non-enzymatic method-I recovered maximum DNA from the fermented bamboo shoot and fish products. Across the food types, the in-house developed methods (III, IV) and the commercial method (VI) showed poor DNA recovery. The DNA recovered by all the methods were of good quality (A260/280 = 1.7–2.3) with no PCR inhibitors. For each food type, the efficiency of DNA recovery strongly depended on the extraction method applied and a single method cannot efficiently extract DNA from all the food types.

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.