16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice.
ABSTRACT: BACKGROUND:Characterisation of the bacterial composition of the gut microbiota is increasingly carried out with a view to establish the role of different bacterial species in causation or prevention of disease. It is thus essential that the methods used to determine the microbial composition are robust. Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning. RESULTS:The bacterial community profile detected in the faeces of infants is highly dependent on the methodology used. Bifidobacteria were the most abundant bacteria detected at 6 weeks in faeces from two initially breast-fed babies using fluorescent in situ hybridisation (FISH), in agreement with data from previous culture-based studies. Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1-V3 regions of 16S rRNA genes prior to subsequent sequence analysis. Bifidobacteria were only well represented among amplified 16S rRNA gene sequences when mechanical disruption (bead-beating) procedures for DNA extraction were employed together with optimised "universal" PCR primers. These primers incorporate degenerate bases at positions where mismatches to bifidobacteria and other bacterial taxa occur. The use of a DNA extraction kit with no bead-beating step resulted in a complete absence of bifidobacteria in the sequence data, even when using the optimised primers. CONCLUSIONS:This work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.
Project description:The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, 'bifidobacteria-optimised' universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.
Project description:In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 10(5) sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.
Project description:DNA extraction and primer choice have a large effect on the observed community structure in all microbial amplicon sequencing analyses. Although the biases are well known, no comprehensive analysis has been conducted in activated sludge communities. In this study we systematically explored the impact of a number of parameters on the observed microbial community: bead beating intensity, primer choice, extracellular DNA removal, and various PCR settings. In total, 176 samples were subjected to 16S rRNA amplicon sequencing, and selected samples were investigated through metagenomics and metatranscriptomics. Quantitative fluorescence in situ hybridization was used as a DNA extraction-independent method for qualitative comparison. In general, an effect on the observed community was found on all parameters tested, although bead beating and primer choice had the largest effect. The effect of bead beating intensity correlated with cell-wall strength as seen by a large increase in DNA from Gram-positive bacteria (up to 400%). However, significant differences were present at lower phylogenetic levels within the same phylum, suggesting that additional factors are at play. The best primer set based on in silico analysis was found to underestimate a number of important bacterial groups. For 16S rRNA gene analysis in activated sludge we recommend using the FastDNA SPIN Kit for Soil with four times the normal bead beating and V1-3 primers.
Project description:BACKGROUND: The microbial community analysis of stools requires optimised and standardised protocols for their collection, homogenisation, microbial disruption and nucleic acid extraction. Here we examined whether different layers of the stool are equally representative of the microbiome. We also studied the effect of stool water content, which typically increases in diarrhoeic samples, and of a microbial disruption method on DNA integrity and, therefore, on providing an unbiased microbial composition analysis. RESULTS: We collected faecal samples from healthy subjects and performed microbial composition analysis by pyrosequencing the V4 region of the 16S rRNA gene. To examine the effect of stool structure, we compared the inner and outer layers of the samples (N?=?8). Both layers presented minor differences in microbial composition and abundance at the species level. These differences did not significantly bias the microbial community specific to an individual. To evaluate the effect of stool water content and bead-beating, we used various volumes of a water-based salt solution and beads of distinct weights before nucleic acid extraction (N?=?4). The different proportions of water did not affect the UniFrac-based clustering of samples from the same subject However, the use or omission of a bead-beating step produced different proportions of Gram-positive and Gram-negative bacteria and significant changes in the UniFrac-based clustering of the samples. CONCLUSION: The degree of hydration and homogenisation of faecal samples do not significantly alter their microbial community composition. However, the use of bead-beating is critical for the proper detection of Gram-positive bacteria such as Blautia and Bifidobacterium.
Project description:The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.
Project description:The objective of this research was to compare the composition of bacterial microbiota associated with the ruminal content (RC), ruminal epithelium (RE) and faeces of Holstein dairy cows. The RC, RE and faecal samples were collected from six Holstein dairy cows when the animals were slaughtered. Community compositions of bacterial 16S rRNA genes from RC, RE and faeces were determined using a MiSeq sequencing platform with bacterial-targeting universal primers 338F and 806R. UniFrac analysis revealed that the bacterial communities of RC, RE and faeces were clearly separated from each other. Statistically significant dissimilarities were observed between RC and faeces (P = 0.002), between RC and RE (P = 0.003), and between RE and faeces (P = 0.001). A assignment of sequences to taxa showed that the abundance of the predominant phyla Bacteroidetes was lower in RE than in RC, while a significant higher (P < 0.01) abundance of Proteobacteria was present in RE than in RC. When compared with the RC, the abundance of Firmicutes and Verrucomicrobia was higher in faeces, and RC contained a greater abundance of Bacteroidetes and Tenericutes. A higher proportions of Butyrivibrio and Campylobacter dominated RE as compared to RC. The faecal microbiota was less diverse than RC and dominated by genera Turicibacter and Clostridium. In general, these findings clearly demonstrated the striking compositional differences among RC, RE and faeces, indicating that bacterial communities are specific and adapted to the harbouring environment.
Project description:Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.
Project description:Amplification and sequencing of 16S amplicons are widely used for profiling the structure of oral microbiota. However, it remains not clear whether and to what degree DNA extraction and targeted 16S rRNA hypervariable regions influence the analysis. Based on a mock community consisting of five oral bacterial species in equal abundance, we compared the 16S amplicon sequencing results on the Illumina MiSeq platform from six frequently employed DNA extraction procedures and three pairs of widely used 16S rRNA hypervariable primers targeting different 16S rRNA regions. Technical reproducibility of selected 16S regions was also assessed. DNA extraction method exerted considerable influence on the observed bacterial diversity while hypervariable regions had a relatively minor effect. Protocols with beads added to the enzyme-mediated DNA extraction reaction produced more accurate bacterial community structure than those without either beads or enzymes. Hypervariable regions targeting V3-V4 and V4-V5 seemed to produce more reproducible results than V1-V3. Neither sequencing batch nor change of operator affected the reproducibility of bacterial diversity profiles. Therefore, DNA extraction strategy and 16S rDNA hypervariable regions both influenced the results of oral microbiota biodiversity profiling, thus should be carefully considered in study design and data interpretation.
Project description:For specific detection of the probiotic Bifidobacterium sp. strain LW420 in infant feces and for rapid quality control of this strain in culture, three strain-specific 16S rRNA gene-targeted primers have been developed. These primers allow specific detection of the organism via PCR. Specificity of the primers was determined in DNA samples isolated from single-strain and mixed cultures of bifidobacteria and in heterogenous fecal samples. The feasibility of this method for use in specific detection of probiotic strains was investigated through addition of Bifidobacterium sp. strain LW420 to infant instant milk formula (IMF) and PCR analyses of bacterial DNA isolated from feces of 17 newborn IMF-fed infants. In feces of all nine babies that had been fed with the probiotic IMF, the strain-specific PCR signal could be detected. No signal was found in feces of the eight infants that had been fed with a nonprobiotic IMF, demonstrating the specificity of the PCR method. All 17 infants developed a major fecal Bifidobacterium population already after 3 days, as determined through genus-specific and strain-specific PCR. Phenotypical screening of Bifidobacterium sp. strain LW420 and analysis of homology of the 16S rRNA gene sequence of this strain with that of other bifidobacteria deposited in databases do not allow positive classification of LW420 among the currently known species of Bifidobacterium.
Project description:The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.