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Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags.

Wu GD, Lewis JD, Hoffmann C, Chen YY, Knight R, Bittinger K, Hwang J, Chen J, Berkowsky R, Nessel L, Li H, Bushman FD - BMC Microbiol. (2010)

Bottom Line: In the weighted analysis considerable variation was associated with the purification methods.Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method.We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gastroenterology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6076 USA. gdwu@mail.med.upenn.edu

ABSTRACT
Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80 degrees C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method.

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Comparison of the relative abundance of different bacterial taxa under the conditions tested using weighted UniFrac. Weighted UniFrac was used to generate a matrix of pairwise distances between communities, then a scatterplot was generated from the matrix of distances using Principal Coordinate Analysis. The same scatterplot is shown in A)-C), but colored by subject A), storage method B), or extraction method C). The P-values cited in the text were generated using distances from the original UniFrac matrix.
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Figure 4: Comparison of the relative abundance of different bacterial taxa under the conditions tested using weighted UniFrac. Weighted UniFrac was used to generate a matrix of pairwise distances between communities, then a scatterplot was generated from the matrix of distances using Principal Coordinate Analysis. The same scatterplot is shown in A)-C), but colored by subject A), storage method B), or extraction method C). The P-values cited in the text were generated using distances from the original UniFrac matrix.

Mentions: The 16S rRNA gene sequence reads from the 57 communities were aligned to generate a phylogenetic tree using FastTree2 [35]. Communities were then compared in a pair-wise fashion by means of the UniFrac distance metric, which quantifies the proportion of the branch length on the tree unique to each community in each pair. Pairwise UniFrac distances were used to generate a matrix of all distances between pairs of communities, and principal coordinate analysis used for the cluster analysis (Figures 3 and 4). All steps were carried out in an automated fashion within QIIME [36]. UniFrac analysis was carried out either unweighted, using only presence-absence information, or weighted, which takes in to account the relative proportions of each group.


Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags.

Wu GD, Lewis JD, Hoffmann C, Chen YY, Knight R, Bittinger K, Hwang J, Chen J, Berkowsky R, Nessel L, Li H, Bushman FD - BMC Microbiol. (2010)

Comparison of the relative abundance of different bacterial taxa under the conditions tested using weighted UniFrac. Weighted UniFrac was used to generate a matrix of pairwise distances between communities, then a scatterplot was generated from the matrix of distances using Principal Coordinate Analysis. The same scatterplot is shown in A)-C), but colored by subject A), storage method B), or extraction method C). The P-values cited in the text were generated using distances from the original UniFrac matrix.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921404&req=5

Figure 4: Comparison of the relative abundance of different bacterial taxa under the conditions tested using weighted UniFrac. Weighted UniFrac was used to generate a matrix of pairwise distances between communities, then a scatterplot was generated from the matrix of distances using Principal Coordinate Analysis. The same scatterplot is shown in A)-C), but colored by subject A), storage method B), or extraction method C). The P-values cited in the text were generated using distances from the original UniFrac matrix.
Mentions: The 16S rRNA gene sequence reads from the 57 communities were aligned to generate a phylogenetic tree using FastTree2 [35]. Communities were then compared in a pair-wise fashion by means of the UniFrac distance metric, which quantifies the proportion of the branch length on the tree unique to each community in each pair. Pairwise UniFrac distances were used to generate a matrix of all distances between pairs of communities, and principal coordinate analysis used for the cluster analysis (Figures 3 and 4). All steps were carried out in an automated fashion within QIIME [36]. UniFrac analysis was carried out either unweighted, using only presence-absence information, or weighted, which takes in to account the relative proportions of each group.

Bottom Line: In the weighted analysis considerable variation was associated with the purification methods.Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method.We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gastroenterology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6076 USA. gdwu@mail.med.upenn.edu

ABSTRACT
Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80 degrees C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method.

Show MeSH
Related in: MedlinePlus