| Title |
Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing
|
|---|---|
| Published in |
Applied Microbiology and Biotechnology, July 2012
|
| DOI | 10.1007/s00253-012-4244-4 |
| Pubmed ID | |
| Authors |
Feng Guo, Tong Zhang |
| Abstract |
Standardization of DNA extraction is a fundamental issue of fidelity and comparability in investigations of environmental microbial communities. Commercial kits for soil or feces are often adopted for studies of activated sludge because of a lack of specific kits, but they have never been evaluated regarding their effectiveness and potential biases based on high throughput sequencing. In this study, seven common DNA extraction kits were evaluated, based on not only yield/purity but also sequencing results, using two activated sludge samples (two sub-samples each, i.e. ethanol-fixed and fresh, as-is). The results indicate that the bead-beating step is necessary for DNA extraction from activated sludge. The two kits without the bead-beating step yielded very low amounts of DNA, and the least abundant operational taxonomic units (OTUs), and significantly underestimated the Gram-positive Actinobacteria, Nitrospirae, Chloroflexi, and Alphaproteobacteria and overestimated Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, and the rare phyla whose cell walls might have been readily broken. Among the other five kits, FastDNA(@) SPIN Kit for Soil extracted the most and the purest DNA. Although the number of total OTUs obtained using this kit was not the highest, the abundant OTUs and abundance of Actinobacteria demonstrated its efficiency. The three MoBio kits and one ZR kit produced fair results, but had a relatively low DNA yield and/or less Actinobacteria-related sequences. Moreover, the 50 % ethanol fixation increased the DNA yield, but did not change the sequenced microbial community in a significant way. Based on the present study, the FastDNA SPIN kit for Soil is recommended for DNA extraction of activated sludge samples. More importantly, the selection of the DNA extraction kit must be done carefully if the samples contain dominant lysing-resistant groups, such as Actinobacteria and Nitrospirae. |
X Demographics
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Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United States | 1 | 100% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 1 | 100% |
Mendeley readers
The data shown below were compiled from readership statistics for 261 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United States | 2 | <1% |
| Germany | 1 | <1% |
| Brazil | 1 | <1% |
| Canada | 1 | <1% |
| United Kingdom | 1 | <1% |
| Spain | 1 | <1% |
| Denmark | 1 | <1% |
| Unknown | 253 | 97% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 62 | 24% |
| Researcher | 50 | 19% |
| Student > Master | 42 | 16% |
| Student > Bachelor | 21 | 8% |
| Student > Doctoral Student | 17 | 7% |
| Other | 28 | 11% |
| Unknown | 41 | 16% |
| Readers by discipline | Count | As % |
|---|---|---|
| Agricultural and Biological Sciences | 81 | 31% |
| Biochemistry, Genetics and Molecular Biology | 41 | 16% |
| Environmental Science | 38 | 15% |
| Engineering | 14 | 5% |
| Immunology and Microbiology | 13 | 5% |
| Other | 17 | 7% |
| Unknown | 57 | 22% |
Attention Score in Context
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#19,611,252
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Outputs from Applied Microbiology and Biotechnology
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Outputs of similar age from Applied Microbiology and Biotechnology
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