Differences in the gut microbiome composition of Korean children and adult samples based on different DNA isolation kits

Abstract

Recent studies have revealed that the composition of human gut microbiota varies according to region, race, age, diet, living environment, and sampling and DNA extraction method. The purpose of this study was to broaden our understanding of the intestinal microbial composition of Koreans by conducting a 16S rRNA amplicon sequencing on 78 Korean samples composed of adults, children, normal and obese groups. We compared the microbiome composition and diversity of these groups at different levels including the phylum and genus level using two different stool DNA extraction kits of QIAamp® PowerFecal® DNA Kit (Qiagen, Hilden, Germany) and CT Max Fecal DNA Kit (Ct bio, Korea). We found that Ct bio (Ct) kit recovered higher DNA yields and OTUs than QIAamp® PowerFecal® DNA Kit (Qia). The Ct kit, which adopted more rigorous bead beating method, detected the most Gram-positive (G+) bacteria, Firmicutes, at the Phylum level, whereas the Qia kit, which used a less rigorous cell lysis method, found the most Gram-negative (G-) bacteria, Bacteroidetes. The Firmicutes-to-Bacteroidetes (F/B) ratio showed no significant difference between the obese and the normal groups of same kit; however, they were significantly different with two different kits. There was a difference in the intestinal flora between healthy Korean adults and children. The taxa that differed significantly between the adults and children were Bacteroides, Bifidobacterium, Prevotella, and Subdoligranulum. There was no significant difference in the intestinal flora between the normal weight group and the obese group in adults and children, respectively. This is probably because the difference in body mass index (BMI) between the sample groups collected in this study is statistically significant, but it is not large enough to show a clear difference in the flora. Therefore, these results should be interpreted with caution while considering the BMI values and Korean obesity criterion together.

Fig 1. Comparison of DNA quality and bacterial diversity using two DNA isolation kits (Ct and Qia).

A. DNA yield. The gray lines represent the same sample pair. B. DNA purity. C. OTUs D. Chao1 index.

Fig 2. Relative abundance of microbiome of adults and children.

A. Variation in bacterial relative abundance at the phylum level with Ct kit. B. Bacterial relative abundance at the phylum level with Qia kit. C. Bacterial relative abundance at the genus level with Ct kit. D. Bacterial relative abundance at the genus level with Qia kit.

Fig 3. Comparison of relative abundance of four major microbiomes at phylum level using Ct and Qia kits.

G+: Gram-positive bacteria. G-: Gram-negative bacteria.

Fig 4. Firmicutes/Bacteroidetes (F/B) ratio of subgroups.

A. F/B ratio distribution between normal vs. obese group. B. F/B ratio distribution between adult vs. children group. C. F/B ratio distribution between Ct and Qia kits in Normal and obese groups, respectively. The gray lines represent the same sample pair. D. F/B ratio distribution between Ct and Qia kits in adults and children groups, respectively.

Fig 5. Beta diversity observation in different subgroups.

A. NMDS plot of normal and obese group microbiome communities with Ct kit. B. NMDS plot of adults and children microbiome communities with Ct kit. C. NMDS plot of normal and obese group microbiome communities with Qia kit. D. NMDS plot of adults and children microbiome communities with Qia kit.

Fig 6. Characterization of microbiomes of adults and children by LEfSe and LDA analysis.

A. Taxa with significant differences in abundance between adults and children with Ct kit. B. Taxa with significant differences in abundance between adults and children with Qia kit. Histogram of log₁₀(LDA scores) for features with differential abundance between adults and children with Ct kit (C) and with Qia kit (D). Taxa of |log₁₀(LDA scores)| >3 are presented.