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16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice.

Walker AW, Martin JC, Scott P, Parkhill J, Flint HJ, Scott KP - Microbiome (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, AB21 9SB UK ; Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA UK.

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.

No MeSH data available.


Related in: MedlinePlus

Comparison of bacterial families detected in faecal samples from two babies. Sequence data is based on 16S rRNA gene amplicons obtained using the 27f-YM (blue) or 27f-Mix (red) forward primers. a Baby N-BF: Data shows the mean percentage of sequences in each bacterial family after 15 separate DNA extractions at seven time points. b Baby C-MF: Data shows the mean percentage of sequences in each bacterial family after six separate DNA extractions at three time points. For both panels, individual data points are plotted as open circles; centre lines in the box plots show the medians; crosses represent sample means; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Plotted using BoxPlotR [52]
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Fig2: Comparison of bacterial families detected in faecal samples from two babies. Sequence data is based on 16S rRNA gene amplicons obtained using the 27f-YM (blue) or 27f-Mix (red) forward primers. a Baby N-BF: Data shows the mean percentage of sequences in each bacterial family after 15 separate DNA extractions at seven time points. b Baby C-MF: Data shows the mean percentage of sequences in each bacterial family after six separate DNA extractions at three time points. For both panels, individual data points are plotted as open circles; centre lines in the box plots show the medians; crosses represent sample means; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Plotted using BoxPlotR [52]

Mentions: The compositional differences observed using the different primer sets were marked (Table 2, Fig. 1). As expected, the pyrosequencing 16S rRNA gene data generated using the primer combinations specific for bifidobacteria (27f-Bif/534r or Bif164f/Bif662r; Table 1) resulted in most of the sequences obtained being derived from Bifidobacterium species. All sequences amplified using the established bifidobacteria-specific primer set (Bif164/Bif662r) corresponded to bifidobacteria, while the 27f-Bif/534r combination was slightly less specific, with 4.5 % of the resulting sequences not identified as bifidobacteria (Table 2). The mixed forward primer set (27f-Mix) picked up on average 30 % more bifidobacteria than the single 27f-YM primer (Table 2), with a proportional reduction in some of the other bacterial genera enumerated. The specific profile generated from different babies clearly shows that the calculated proportional abundance of bifidobacteria depends not only on inter-individual variation but also crucially on primer choice. The use of the mixed forward primer (27f-Mix) significantly increased (at least doubling) the proportion of bifidobacteria detected compared to the single “universal” primer 27f-YM, while there was little difference in the detection of other bacterial genera (Fig. 2). Comparing the average proportional abundance of bifidobacteria in both babies at all ten time points, detected using the two primer sets, revealed that significantly fewer bifidobacteria sequences were detected with the basic primer 27f-YM (p < 0.001).Fig. 2


16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice.

Walker AW, Martin JC, Scott P, Parkhill J, Flint HJ, Scott KP - Microbiome (2015)

Comparison of bacterial families detected in faecal samples from two babies. Sequence data is based on 16S rRNA gene amplicons obtained using the 27f-YM (blue) or 27f-Mix (red) forward primers. a Baby N-BF: Data shows the mean percentage of sequences in each bacterial family after 15 separate DNA extractions at seven time points. b Baby C-MF: Data shows the mean percentage of sequences in each bacterial family after six separate DNA extractions at three time points. For both panels, individual data points are plotted as open circles; centre lines in the box plots show the medians; crosses represent sample means; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Plotted using BoxPlotR [52]
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482049&req=5

Fig2: Comparison of bacterial families detected in faecal samples from two babies. Sequence data is based on 16S rRNA gene amplicons obtained using the 27f-YM (blue) or 27f-Mix (red) forward primers. a Baby N-BF: Data shows the mean percentage of sequences in each bacterial family after 15 separate DNA extractions at seven time points. b Baby C-MF: Data shows the mean percentage of sequences in each bacterial family after six separate DNA extractions at three time points. For both panels, individual data points are plotted as open circles; centre lines in the box plots show the medians; crosses represent sample means; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Plotted using BoxPlotR [52]
Mentions: The compositional differences observed using the different primer sets were marked (Table 2, Fig. 1). As expected, the pyrosequencing 16S rRNA gene data generated using the primer combinations specific for bifidobacteria (27f-Bif/534r or Bif164f/Bif662r; Table 1) resulted in most of the sequences obtained being derived from Bifidobacterium species. All sequences amplified using the established bifidobacteria-specific primer set (Bif164/Bif662r) corresponded to bifidobacteria, while the 27f-Bif/534r combination was slightly less specific, with 4.5 % of the resulting sequences not identified as bifidobacteria (Table 2). The mixed forward primer set (27f-Mix) picked up on average 30 % more bifidobacteria than the single 27f-YM primer (Table 2), with a proportional reduction in some of the other bacterial genera enumerated. The specific profile generated from different babies clearly shows that the calculated proportional abundance of bifidobacteria depends not only on inter-individual variation but also crucially on primer choice. The use of the mixed forward primer (27f-Mix) significantly increased (at least doubling) the proportion of bifidobacteria detected compared to the single “universal” primer 27f-YM, while there was little difference in the detection of other bacterial genera (Fig. 2). Comparing the average proportional abundance of bifidobacteria in both babies at all ten time points, detected using the two primer sets, revealed that significantly fewer bifidobacteria sequences were detected with the basic primer 27f-YM (p < 0.001).Fig. 2

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, AB21 9SB UK ; Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA UK.

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.

No MeSH data available.


Related in: MedlinePlus