Limits...
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.

Show MeSH

Related in: MedlinePlus

Composition of the gut microbiome in the ten subjects studied. Bacterial taxonomic assignments are indicated to the right of the heat map at the Phylum and Genus level except in cases where small numbers were detected (e. g. Proteobacteria), in which case taxa are summarized at higher levels. The relative abundance of each bacterial group is color coded as indicated by the key on the left (the number beside each colored tile indicates the lower bound for the indicated interval). Two samples were compared for each stool specimen, sampled on cm a part but otherwise worked up identically (conditions 1 and 2 in Table 2). The numbers of reads for the two samples from each subject were compared for significant differences using Fisher's exact test. The * indicates P < 0.05. Note that because each sequence read is treated as an individual measurement, the sample size is very large, with the result that many taxa with only modest differences nevertheless achieve significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2921404&req=5

Figure 1: Composition of the gut microbiome in the ten subjects studied. Bacterial taxonomic assignments are indicated to the right of the heat map at the Phylum and Genus level except in cases where small numbers were detected (e. g. Proteobacteria), in which case taxa are summarized at higher levels. The relative abundance of each bacterial group is color coded as indicated by the key on the left (the number beside each colored tile indicates the lower bound for the indicated interval). Two samples were compared for each stool specimen, sampled on cm a part but otherwise worked up identically (conditions 1 and 2 in Table 2). The numbers of reads for the two samples from each subject were compared for significant differences using Fisher's exact test. The * indicates P < 0.05. Note that because each sequence read is treated as an individual measurement, the sample size is very large, with the result that many taxa with only modest differences nevertheless achieve significance.

Mentions: Figure 1 shows the bacterial taxa detected summarized as a heat map. The most abundant genera are shown together with their Phylum-level assignments. For each subject, two identically processed samples taken 1 cm apart are shown (methods 1 and 2 in Table 2). Overall there is good reproducibility between the two adjacent "gold standard" samples--of the taxa present as greater than 1% of the total, all were detected in the paired sample. However, low abundance taxa were detected sporadically--of the samples present at 0.2%-0.4% of the total in one replicate (red in Figure 1), 35% were not detected in the second replicate. Statistical tests for significant differences are described below.


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)

Composition of the gut microbiome in the ten subjects studied. Bacterial taxonomic assignments are indicated to the right of the heat map at the Phylum and Genus level except in cases where small numbers were detected (e. g. Proteobacteria), in which case taxa are summarized at higher levels. The relative abundance of each bacterial group is color coded as indicated by the key on the left (the number beside each colored tile indicates the lower bound for the indicated interval). Two samples were compared for each stool specimen, sampled on cm a part but otherwise worked up identically (conditions 1 and 2 in Table 2). The numbers of reads for the two samples from each subject were compared for significant differences using Fisher's exact test. The * indicates P < 0.05. Note that because each sequence read is treated as an individual measurement, the sample size is very large, with the result that many taxa with only modest differences nevertheless achieve significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Composition of the gut microbiome in the ten subjects studied. Bacterial taxonomic assignments are indicated to the right of the heat map at the Phylum and Genus level except in cases where small numbers were detected (e. g. Proteobacteria), in which case taxa are summarized at higher levels. The relative abundance of each bacterial group is color coded as indicated by the key on the left (the number beside each colored tile indicates the lower bound for the indicated interval). Two samples were compared for each stool specimen, sampled on cm a part but otherwise worked up identically (conditions 1 and 2 in Table 2). The numbers of reads for the two samples from each subject were compared for significant differences using Fisher's exact test. The * indicates P < 0.05. Note that because each sequence read is treated as an individual measurement, the sample size is very large, with the result that many taxa with only modest differences nevertheless achieve significance.
Mentions: Figure 1 shows the bacterial taxa detected summarized as a heat map. The most abundant genera are shown together with their Phylum-level assignments. For each subject, two identically processed samples taken 1 cm apart are shown (methods 1 and 2 in Table 2). Overall there is good reproducibility between the two adjacent "gold standard" samples--of the taxa present as greater than 1% of the total, all were detected in the paired sample. However, low abundance taxa were detected sporadically--of the samples present at 0.2%-0.4% of the total in one replicate (red in Figure 1), 35% were not detected in the second replicate. Statistical tests for significant differences are described below.

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