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


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2010-07-30

AUTHORS

Gary D Wu, James D Lewis, Christian Hoffmann, Ying-Yu Chen, Rob Knight, Kyle Bittinger, Jennifer Hwang, Jun Chen, Ronald Berkowsky, Lisa Nessel, Hongzhe Li, Frederic D Bushman

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°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. More... »

PAGES

206

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1471-2180-10-206

DOI

http://dx.doi.org/10.1186/1471-2180-10-206

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1027026649

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/20673359


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29 schema:description 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°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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36 DNA purification methods
37 Firmicutes sequences
38 abundance
39 account
40 acquisition
41 amplification
42 analysis
43 analytical procedure
44 approach
45 bacterial communities
46 bacterial taxa
47 bead beating
48 beating
49 center
50 commercial kits
51 community
52 comparison
53 considerable variation
54 convenience
55 development
56 different analytical procedures
57 different methodologies
58 disease
59 effect
60 experimental convenience
61 fecal samples
62 feces
63 findings
64 freezing
65 gene segments
66 gene tags
67 gut
68 gut microbiome
69 health
70 hot phenol
71 hot phenol method
72 hours
73 human feces
74 human gut
75 human gut microbiome
76 ice
77 immediate freezing
78 individuals
79 information
80 interest centers
81 kit
82 little variation
83 lysis method
84 major points
85 method
86 methodology
87 microbiome
88 one
89 optimal method
90 phenol
91 phenol method
92 point
93 presence-absence information
94 procedure
95 process
96 protocol
97 purification
98 purification method
99 pyrosequencing
100 rDNA
101 rRNA gene tags
102 recommendations
103 recovery
104 relative abundance
105 reproducibility
106 role
107 sample one
108 samples
109 segments
110 sequence
111 sequence acquisition
112 sequence tags
113 sequencing methods
114 single stool specimen
115 specimen
116 stool specimen
117 storage
118 storage methods
119 study
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121 survey
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123 taxa
124 unweighted analysis
125 use
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