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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

Dendrogram illustrating the microbial composition in two babies, pre-weaning. Thirty-eight sequenced samples are shown, derived from DNA extracted using the Fast DNA SPIN Kit for Soil, which contains a bead-beating step, from nine distinct samples from two babies at different time points, amplified with four primer sets (Table 2), and a further single DNA extraction of one sample using the, non-bead-beating, Qiagen QIAamp kit. N-BF indicates samples from the natural birth, solely breast-fed infant. C-MF indicates samples from the C-section birth, mixed-feeding infant. The infant age at time of sampling is shown (in weeks). The dendrogram clearly shows the difference in composition, specifically the lack of bifidobacterial sequences, between the Qiagen kit (marked with QIA and red branches in the figure) and every other sample. Different PCR primer combinations are indicated by branch colouring: yellow—27f-YM primer; green—27f-Mix combination of forward primers; the two shades of blue represent samples processed with the 27f-Bif and Bif164 control primer sets. Adjacent bar charts show the bacterial composition of the sequence data at the family level. Using the 27f-Mix PCR primers increased detection of bifidobacterial sequences compared to using the 27f-YM primer, which has two mismatches to the Bifidobacterium genus
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Fig1: Dendrogram illustrating the microbial composition in two babies, pre-weaning. Thirty-eight sequenced samples are shown, derived from DNA extracted using the Fast DNA SPIN Kit for Soil, which contains a bead-beating step, from nine distinct samples from two babies at different time points, amplified with four primer sets (Table 2), and a further single DNA extraction of one sample using the, non-bead-beating, Qiagen QIAamp kit. N-BF indicates samples from the natural birth, solely breast-fed infant. C-MF indicates samples from the C-section birth, mixed-feeding infant. The infant age at time of sampling is shown (in weeks). The dendrogram clearly shows the difference in composition, specifically the lack of bifidobacterial sequences, between the Qiagen kit (marked with QIA and red branches in the figure) and every other sample. Different PCR primer combinations are indicated by branch colouring: yellow—27f-YM primer; green—27f-Mix combination of forward primers; the two shades of blue represent samples processed with the 27f-Bif and Bif164 control primer sets. Adjacent bar charts show the bacterial composition of the sequence data at the family level. Using the 27f-Mix PCR primers increased detection of bifidobacterial sequences compared to using the 27f-YM primer, which has two mismatches to the Bifidobacterium genus

Mentions: In order to compare the effect of storage and DNA extraction method, DNA was extracted from a frozen aliquot of one faecal sample using both the FastDNA SPIN Kit for Soil (2-min bead-beating time; method 1) and the QIAamp DNA stool mini kit, which does not include a mechanical disruption step, following the manufacturer’s instructions (method 2). Using extraction method 1, the thawed faecal sample (stored frozen at −20 °C for 3 months) gave the same proportion of bifidobacteria as the original non-frozen, freshly extracted sample (Additional file 1: Figure S3). However, despite being the dominant genus when processed using method 1, no bifidobacteria were detected in the aliquot of this frozen sample when DNA was extracted using method 2 (the QIAamp DNA stool mini kit), regardless of which “universal” PCR primer sets were subsequently employed (Fig. 1). Indeed, the dominant bacterial family in the DNA sample extracted using method 2 was Veillonellaceae, comprising 40–50 % of the total bacteria detected, despite being a minor component (<5 %) in samples extracted using method 1 (Additional file 1: Figure S3). Lactobacillaceae were also undetectable when method 2 was used for DNA extraction despite otherwise comprising >25 % of the bacterial composition when DNA was extracted using method 1 prior to 16S rRNA gene sequence analysis. The relative increase in abundance of Lactobacillaceae in the sample that had been stored frozen was countered by a decrease in Lachnospiraceae. The choice of DNA extraction method therefore had a much greater effect on the apparent microbiota composition than did storage of the sample for 3 months at −20 °C prior to DNA extraction. Effective recovery of bifidobacterial sequences depends on the DNA extraction process incorporating a bead-beating step. The QIAamp kit, employed following the manufacturer’s extraction protocol, was clearly inadequate for extracting DNA from faecal samples for the purpose of profiling the total bacterial community using 16S rRNA gene sequencing.Fig. 1


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)

Dendrogram illustrating the microbial composition in two babies, pre-weaning. Thirty-eight sequenced samples are shown, derived from DNA extracted using the Fast DNA SPIN Kit for Soil, which contains a bead-beating step, from nine distinct samples from two babies at different time points, amplified with four primer sets (Table 2), and a further single DNA extraction of one sample using the, non-bead-beating, Qiagen QIAamp kit. N-BF indicates samples from the natural birth, solely breast-fed infant. C-MF indicates samples from the C-section birth, mixed-feeding infant. The infant age at time of sampling is shown (in weeks). The dendrogram clearly shows the difference in composition, specifically the lack of bifidobacterial sequences, between the Qiagen kit (marked with QIA and red branches in the figure) and every other sample. Different PCR primer combinations are indicated by branch colouring: yellow—27f-YM primer; green—27f-Mix combination of forward primers; the two shades of blue represent samples processed with the 27f-Bif and Bif164 control primer sets. Adjacent bar charts show the bacterial composition of the sequence data at the family level. Using the 27f-Mix PCR primers increased detection of bifidobacterial sequences compared to using the 27f-YM primer, which has two mismatches to the Bifidobacterium genus
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Dendrogram illustrating the microbial composition in two babies, pre-weaning. Thirty-eight sequenced samples are shown, derived from DNA extracted using the Fast DNA SPIN Kit for Soil, which contains a bead-beating step, from nine distinct samples from two babies at different time points, amplified with four primer sets (Table 2), and a further single DNA extraction of one sample using the, non-bead-beating, Qiagen QIAamp kit. N-BF indicates samples from the natural birth, solely breast-fed infant. C-MF indicates samples from the C-section birth, mixed-feeding infant. The infant age at time of sampling is shown (in weeks). The dendrogram clearly shows the difference in composition, specifically the lack of bifidobacterial sequences, between the Qiagen kit (marked with QIA and red branches in the figure) and every other sample. Different PCR primer combinations are indicated by branch colouring: yellow—27f-YM primer; green—27f-Mix combination of forward primers; the two shades of blue represent samples processed with the 27f-Bif and Bif164 control primer sets. Adjacent bar charts show the bacterial composition of the sequence data at the family level. Using the 27f-Mix PCR primers increased detection of bifidobacterial sequences compared to using the 27f-YM primer, which has two mismatches to the Bifidobacterium genus
Mentions: In order to compare the effect of storage and DNA extraction method, DNA was extracted from a frozen aliquot of one faecal sample using both the FastDNA SPIN Kit for Soil (2-min bead-beating time; method 1) and the QIAamp DNA stool mini kit, which does not include a mechanical disruption step, following the manufacturer’s instructions (method 2). Using extraction method 1, the thawed faecal sample (stored frozen at −20 °C for 3 months) gave the same proportion of bifidobacteria as the original non-frozen, freshly extracted sample (Additional file 1: Figure S3). However, despite being the dominant genus when processed using method 1, no bifidobacteria were detected in the aliquot of this frozen sample when DNA was extracted using method 2 (the QIAamp DNA stool mini kit), regardless of which “universal” PCR primer sets were subsequently employed (Fig. 1). Indeed, the dominant bacterial family in the DNA sample extracted using method 2 was Veillonellaceae, comprising 40–50 % of the total bacteria detected, despite being a minor component (<5 %) in samples extracted using method 1 (Additional file 1: Figure S3). Lactobacillaceae were also undetectable when method 2 was used for DNA extraction despite otherwise comprising >25 % of the bacterial composition when DNA was extracted using method 1 prior to 16S rRNA gene sequence analysis. The relative increase in abundance of Lactobacillaceae in the sample that had been stored frozen was countered by a decrease in Lachnospiraceae. The choice of DNA extraction method therefore had a much greater effect on the apparent microbiota composition than did storage of the sample for 3 months at −20 °C prior to DNA extraction. Effective recovery of bifidobacterial sequences depends on the DNA extraction process incorporating a bead-beating step. The QIAamp kit, employed following the manufacturer’s extraction protocol, was clearly inadequate for extracting DNA from faecal samples for the purpose of profiling the total bacterial community using 16S rRNA gene sequencing.Fig. 1

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