Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing

Guo, Feng; Zhang, Tong

doi:10.1007/s00253-012-4244-4

Cited by 145 publications

(128 citation statements)

References 34 publications

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“…For factors affecting on bacterial consortia profiling by high throughput sequencing, current methodological investigations mainly focused on DNA extraction [31] and primer coverage [29,36]. The shortages from short read length are not often mentioned partially because the technique unprecedentedly refined the resolution of community profile and quantitative capability.…”

Section: Discussionmentioning

confidence: 99%

“…We confirm that: i) the sampling sites were not privately-owned or protected in any way; and ii) the field studies did not involve endangered or protected species. This kit has been found to be the most efficient method for extracting DNA from activated sludge [31]. Equal masses of DNA from different samples were mixed for the following PCR amplification.…”

Section: Modified Greengenes Database Using Validated Sequences Of Thmentioning

confidence: 99%

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Taxonomic Precision of Different Hypervariable Regions of 16S rRNA Gene and Annotation Methods for Functional Bacterial Groups in Biological Wastewater Treatment

Guo¹,

Ju²,

Lin³

et al. 2015

Bioremediation of Wastewater

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High throughput sequencing of 16S rRNA gene leads us into a deeper understanding on bacterial diversity for complex environmental samples, but introduces blurring due to the relatively low taxonomic capability of short read. For wastewater treatment plant, only those functional bacterial genera categorized as nutrient remediators, bulk/foaming species, and potential pathogens are significant to biological wastewater treatment and environmental impacts. Precise taxonomic assignment of these bacteria at least at genus level is important for microbial ecological research and routine wastewater treatment monitoring. Therefore, the focus of this study was to evaluate the taxonomic precisions of different ribosomal RNA (rRNA) gene hypervariable regions generated from a mix activated sludge sample. In addition, three commonly used classification methods including RDP Classifier, BLAST-based best-hit annotation, and the lowest common ancestor annotation by MEGAN were evaluated by comparing their consistency. Under an unsupervised way, analysis of consistency among different classification methods suggests there are no hypervariable regions with good taxonomic coverage for all genera. Taxonomic assignment based on certain regions of the 16S rRNA genes, e.g. the V1&V2 regions -provide fairly consistent taxonomic assignment for a relatively wide range of genera. Hence, it is recommended to use these regions for studying functional groups in activated sludge. Moreover, the inconsistency among methods also demonstrated that a specific method might not be suitable for identification of some bacterial genera using certain 16S rRNA gene regions. As a general rule, drawing conclusions based only on one sequencing region and one classification method should be avoided due to the potential false negative results.

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Section: Discussionmentioning

confidence: 99%

Section: Modified Greengenes Database Using Validated Sequences Of Thmentioning

confidence: 99%

Taxonomic Precision of Different Hypervariable Regions of 16S rRNA Gene and Annotation Methods for Functional Bacterial Groups in Biological Wastewater Treatment

Guo¹,

Ju²,

Lin³

et al. 2015

Bioremediation of Wastewater

Self Cite

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“…Hence, microbes with cell walls that are difficult to lyse will effectively seem less abundant if sub-optimal extraction protocols are used (Bollet et al, 1991;Filippidou et al, 2015). Furthermore, activated sludge samples contain various chemicals that render some techniques unsuccessful due to inhibition (Guo and Zhang, 2013). Thus, the method for DNA extraction needs to be robust in order to cope with the challenges presented by activated sludge.…”

Section: General Considerationsmentioning

confidence: 99%

“…However, despite much research effort into different DNA extraction protocols, it seems unlikely that there will ever be a perfect protocol. The biases introduced in DNA extraction can only be minimised not circumvented (Guo and Zhang, 2013;Albertsen et al, 2015). The aim of this section is to give a brief overview of the steps involved in DNA extraction and to make general recommendations when working with activated sludge.…”

Section: General Considerationsmentioning

confidence: 99%

“…Ideally, biological replicates are stored to ensure that sample variance can be analysed and that extra biomass is available in case something goes wrong. It is important to minimise the time from sampling to freezing as the changed conditions outside the original environment might favour the growth of some species over others, rendering the sample unsuitable for comparative analysis (Guo and Zhang, 2013). Sampling should preferably take place quite often, e.g.…”

Section: Samplingmentioning

confidence: 99%

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Experimental Methods in Wastewater Treatment

Loosdrecht¹,

Nielsen²,

López-Vázquez³

et al. 2016

Water Intelligence Online

137

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PrefaceWastewater treatment is a core technology for water resources protection and reuse, as is clearly demonstrated by the great success of its consequent implementation in many countries worldwide. During the last decennia scientific research has made vast progress in understanding the complex and interdisciplinary aspects of the biological, biochemical, chemical and mechanical processes involved. It can be concluded that the global application of existing knowledge and experience in wastewater treatment technology will represent a cornerstone in future water management, as expressed in the Strategic Development Goals accepted by the UN in September 2015.Only about one fifth of the wastewater produced globally is currently being adequately treated. To achieve the goal for sustainable water management by 2030 would require extra wastewater treatment facilities for about 600,000 people each day. I am convinced that this book will make its own significant contribution to meeting this ambitious goal.In the near future, most of the global population will live in cities and in low and middle-income countries, where most wastewater is not adequately treated. Probably the most limiting factor in achieving the goals for sustainable water management is the lack of qualified, well-trained professionals, able to comprehend the scientific research results and transfer them into practice. It is therefore of prime importance to make currently available scientific advances and proven experiences in wastewater treatment technology applications easily accessible worldwide. This was one of the drivers for the development of this book, which represents an innovative contribution to help overcome such a capacity development challenge. The book is most definitely expected to contribute to bridging the gaps between the science and technology, and their practical applications.The great collection of authors and reviewers represents an interdisciplinary team of globally acknowledged experts. The book will therefore make a major contribution to establishing a common professional language, enhancing global communication between wastewater professionals. In addition, the authors have linked the description of the scientific basis for wastewater treatment processes with a video-based online course for the training of students, researchers, engineers, laboratory technicians and treatment plant operators, demonstrating commonly accepted experimentation procedures and their application for lab-, pilot-, and full-scale treatment plant operation.From the perspective of the IWA this book also has the great potential to enhance the development of a new generation of researchers and enable them to communicate on a global scale and beyond their specific field of expertise. Both aspects are urgently needed to develop adapted solutions for specific local conditions and to make them globally available for implementation.There has been a trend for some time that scientific research and practice have been growing apart from each other. Part of the rea...

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Metagenomic Approaches for Antibiotic Resistance Gene Detection in Wastewater Treatment Plants

Yang

Zhang²

2017

Antimicrobial Resistance in Wastewater Treatment Processes

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Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing

Cited by 145 publications

References 34 publications

Taxonomic Precision of Different Hypervariable Regions of 16S rRNA Gene and Annotation Methods for Functional Bacterial Groups in Biological Wastewater Treatment

Taxonomic Precision of Different Hypervariable Regions of 16S rRNA Gene and Annotation Methods for Functional Bacterial Groups in Biological Wastewater Treatment

Experimental Methods in Wastewater Treatment

Metagenomic Approaches for Antibiotic Resistance Gene Detection in Wastewater Treatment Plants

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