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Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis.

ABSTRACT: To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6-24?h, before transfer and storage at -80?°C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4?°C within 24?h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals.

SUBMITTER: Penington JS

PROVIDER: S-EPMC5847573 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis.

Penington Jocelyn Sietsma JS Penno Megan A S MAS Ngui Katrina M KM Ajami Nadim J NJ Roth-Schulze Alexandra J AJ Wilcox Stephen A SA Bandala-Sanchez Esther E Wentworth John M JM Barry Simon C SC Brown Cheryl Y CY Couper Jennifer J JJ Petrosino Joseph F JF Papenfuss Anthony T AT Harrison Leonard C LC

Scientific reports 20180312 1

To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6-24 h, before transfer and storage at -80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extra ...[more]

PMID: 29531234

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Project description:BackgroundObesity is a global epidemic in the industrialized and developing world, and many children suffer from obesity-related complications. Gut microbiota dysbiosis might have significant effect on the development of obesity. The microbiota continues to develop through childhood and thus childhood may be the prime time for microbiota interventions to realize health promotion or disease prevention. Therefore, it is crucial to understand the structure and function of pediatric gut microbiota.MethodsAccording to the inclusion criteria and exclusion criteria, twenty-three normal weight and twenty-eight obese children were recruited from Nanjing, China. Genomic DNA was extracted from fecal samples. The V4 region of the bacterial 16S rDNA was amplified by PCR, and sequencing was applied to analyze the gut microbiota diversity and composition using the Illumina HiSeq 2500 platform.ResultsThe number of operational taxonomic units (OTUs) showed a decrease in the diversity of gut microbiota with increasing body weight. The alpha diversity indices showed that the normal weight group had higher abundance and observed species than the obese group (Chao1: P < 0.001; observed species: P < 0.001; PD whole tree: P < 0.001; Shannon index: P = 0.008). Principal coordinate analysis (PCoA) and Nonmetric multidimensional scaling (NMDS) revealed significant differences in gut microbial community structure between the normal weight group and the obese group. The liner discriminant analysis (LDA) effect size (LEfSe) analysis showed that fifty-five species of bacteria were abundant in the fecal samples of the normal weight group and forty-five species of bacteria were abundant in the obese group. In regard to phyla, the gut microbiota in the obese group had lower proportions of Bacteroidetes (51.35%) compared to the normal weight group (55.48%) (P = 0.030). There was no statistical difference in Firmicutes between the two groups (P = 0.436), and the Firmicutes/Bacteroidetes between the two groups had no statistical difference (P = 0.983). At the genus level, Faecalibacterium, Phascolarctobacterium, Lachnospira, Megamonas, and Haemophilus were significantly more abundant in the obese group than in the normal weight group (P = 0.048, P = 0.018, P < 0.001, P = 0.040, and P = 0.003, respectively). The fecal microbiota of children in the obese group had lower proportions of Oscillospira and Dialister compared to the normal weight group (P = 0.002 and P = 0.002, respectively).ConclusionsOur results showed a decrease in gut microbiota abundance and diversity as the BMI increased. Variations in the bacterial community structure were associated with obesity. Gut microbiota dysbiosis might play a crucial part in the development of obesity in Chinese children.

Dataset Information

Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis.

Publications

Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis.

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