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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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Analysis of recovery efficiency after 454/Roche GS FLX sequencing of a cloned DNA mock community. A) Bar graph illustrating proportional recovery of 16S rRNA gene pyrosequence reads from a plasmid DNA mock community. A total of 28,161 sequence reads were used for this analysis (Additional File 4). Each of the 10 templates consisted of a bacterial 16S rRNA gene sequence cloned in a bacterial plasmid. "Even mix" indicates that the same copy number for each of the 10 templates was used in the amplification reaction. "Staggered mix" indicates different amounts. The "Staggered mix 2" sample was amplified with a different polymerase mixture (Promega's GreenTaq Master Mix, Madison, WI) instead of AmpliTaq which was used in all other experiments, revealing that the two mixtures yielded similar results. The taxonomic assignments in this and subsequent figures are color coded as indicated. B) Scatter plot comparing the theoretical proportion of each input sequences (x-axis) to the proportions inferred from 454 GS FLX sequence data (y-axis).
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Figure 6: Analysis of recovery efficiency after 454/Roche GS FLX sequencing of a cloned DNA mock community. A) Bar graph illustrating proportional recovery of 16S rRNA gene pyrosequence reads from a plasmid DNA mock community. A total of 28,161 sequence reads were used for this analysis (Additional File 4). Each of the 10 templates consisted of a bacterial 16S rRNA gene sequence cloned in a bacterial plasmid. "Even mix" indicates that the same copy number for each of the 10 templates was used in the amplification reaction. "Staggered mix" indicates different amounts. The "Staggered mix 2" sample was amplified with a different polymerase mixture (Promega's GreenTaq Master Mix, Madison, WI) instead of AmpliTaq which was used in all other experiments, revealing that the two mixtures yielded similar results. The taxonomic assignments in this and subsequent figures are color coded as indicated. B) Scatter plot comparing the theoretical proportion of each input sequences (x-axis) to the proportions inferred from 454 GS FLX sequence data (y-axis).

Mentions: Figure 6A shows the distribution of sequences. In the even mixture, all ten sequences were recovered in roughly equal proportions, and the staggered communities showed differential recoveries in the expected directions. The two staggered mock communities were sequenced after amplification with two different DNA polymerase mixtures (GreenTaq and AmpliTaq), which did not result in major differences. Figure 6B compares the input and observed proportions for both the even and staggered communities, showing that recovery was close to the input proportions (P < 0.0001). Thus we conclude that the pyrosequencing procedure used here resulted in proportions of sequence reads that closely matched the known input when cloned 16S rRNA genes are used as PCR templates.


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)

Analysis of recovery efficiency after 454/Roche GS FLX sequencing of a cloned DNA mock community. A) Bar graph illustrating proportional recovery of 16S rRNA gene pyrosequence reads from a plasmid DNA mock community. A total of 28,161 sequence reads were used for this analysis (Additional File 4). Each of the 10 templates consisted of a bacterial 16S rRNA gene sequence cloned in a bacterial plasmid. "Even mix" indicates that the same copy number for each of the 10 templates was used in the amplification reaction. "Staggered mix" indicates different amounts. The "Staggered mix 2" sample was amplified with a different polymerase mixture (Promega's GreenTaq Master Mix, Madison, WI) instead of AmpliTaq which was used in all other experiments, revealing that the two mixtures yielded similar results. The taxonomic assignments in this and subsequent figures are color coded as indicated. B) Scatter plot comparing the theoretical proportion of each input sequences (x-axis) to the proportions inferred from 454 GS FLX sequence data (y-axis).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Analysis of recovery efficiency after 454/Roche GS FLX sequencing of a cloned DNA mock community. A) Bar graph illustrating proportional recovery of 16S rRNA gene pyrosequence reads from a plasmid DNA mock community. A total of 28,161 sequence reads were used for this analysis (Additional File 4). Each of the 10 templates consisted of a bacterial 16S rRNA gene sequence cloned in a bacterial plasmid. "Even mix" indicates that the same copy number for each of the 10 templates was used in the amplification reaction. "Staggered mix" indicates different amounts. The "Staggered mix 2" sample was amplified with a different polymerase mixture (Promega's GreenTaq Master Mix, Madison, WI) instead of AmpliTaq which was used in all other experiments, revealing that the two mixtures yielded similar results. The taxonomic assignments in this and subsequent figures are color coded as indicated. B) Scatter plot comparing the theoretical proportion of each input sequences (x-axis) to the proportions inferred from 454 GS FLX sequence data (y-axis).
Mentions: Figure 6A shows the distribution of sequences. In the even mixture, all ten sequences were recovered in roughly equal proportions, and the staggered communities showed differential recoveries in the expected directions. The two staggered mock communities were sequenced after amplification with two different DNA polymerase mixtures (GreenTaq and AmpliTaq), which did not result in major differences. Figure 6B compares the input and observed proportions for both the even and staggered communities, showing that recovery was close to the input proportions (P < 0.0001). Thus we conclude that the pyrosequencing procedure used here resulted in proportions of sequence reads that closely matched the known input when cloned 16S rRNA genes are used as PCR templates.

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