scholarly journals Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9973
Author(s):  
Susheel Bhanu Busi ◽  
Paraskevi Pramateftaki ◽  
Jade Brandani ◽  
Stilianos Fodelianakis ◽  
Hannes Peter ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Extreme Environments ◽  
Extraction Methods ◽  
Ecosystem Functions ◽  
High Mountain ◽  
Acid Extraction ◽  
Microbial Life ◽  
Microbial Biofilms ◽  
Taxonomic Profile

Glacier-fed streams (GFS) are harsh ecosystems dominated by microbial life organized in benthic biofilms, yet the biodiversity and ecosystem functions provided by these communities remain under-appreciated. To better understand the microbial processes and communities contributing to GFS ecosystems, it is necessary to leverage high throughput sequencing. Low biomass and high inorganic particle load in GFS sediment samples may affect nucleic acid extraction efficiency using extraction methods tailored to other extreme environments such as deep-sea sediments. Here, we benchmarked the utility and efficacy of four extraction protocols, including an up-scaled phenol-chloroform protocol. We found that established protocols for comparable sample types consistently failed to yield sufficient high-quality DNA, delineating the extreme character of GFS. The methods differed in the success of downstream applications such as library preparation and sequencing. An adapted phenol-chloroform-based extraction method resulted in higher yields and better recovered the expected taxonomic profile and abundance of reconstructed genomes when compared to commercially-available methods. Affordable and straight-forward, this method consistently recapitulated the abundance and genomes of a mock community, including eukaryotes. Moreover, by increasing the amount of input sediment, the protocol is readily adjustable to the microbial load of the processed samples without compromising protocol efficiency. Our study provides a first systematic and extensive analysis of the different options for extraction of nucleic acids from glacier-fed streams for high-throughput sequencing applications, which may be applied to other extreme environments.

scholarly journals Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams

2020 ◽  
Author(s):  
Susheel Bhanu Busi ◽  
Paraskevi Pramateftaki ◽  
Jade Brandani ◽  
Stylianos Fodelianakis ◽  
Hannes Peter ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Extreme Environments ◽  
Extraction Methods ◽  
Ecosystem Functions ◽  
High Mountain ◽  
Acid Extraction ◽  
Microbial Life ◽  
Microbial Biofilms ◽  
Taxonomic Profile

AbstractGlacier-fed streams (GFS) are harsh ecosystems dominated by microbial life organized in benthic biofilms, yet the biodiversity and ecosystem functions provided by these communities remain under-appreciated. To better understand the microbial processes and communities contributing to GFS ecology, it is necessary to leverage high throughput sequencing. Low biomass and high inorganic particle load in GFS sediment samples may affect nucleic acid extraction efficiency using extraction methods tailored to other extreme environments such as deep-sea sediments. Here, we benchmarked the utility and efficacy of four extraction protocols, including an up-scaled phenol-chloroform protocol. We found that established protocols for comparable sample types consistently failed to yield sufficient high-quality DNA, delineating the extreme character of GFS. The methods differed in the success of downstream applications such as library preparation and sequencing. An adapted phenol-chloroform-based extraction method resulted in higher yields and better recovered the expected taxonomic profile and abundance of reconstructed genomes when compared to commercially-available methods. Affordable and straight-forward, this method consistently recapitulated the abundance and genomes of a “mock” community, including eukaryotes. Moreover, by increasing the amount of input sediment, the protocol is readily adjustable to the microbial load of the processed samples without compromising protocol efficiency. Our study provides a first systematic and extensive analysis of the different options for extraction of nucleic acids from glacier-fed streams for high-throughput sequencing applications, which may be applied to other extreme environments.

scholarly journals Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 566 ◽  
Author(s):  
Siemon Ng ◽  
Cassandra Braxton ◽  
Marc Eloit ◽  
Szi Feng ◽  
Romain Fragnoud ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Nucleic Acids ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Virus Detection ◽  
Extraction Methods ◽  
Control Measures ◽  
Acid Extraction ◽  
Library Preparation ◽  
Sample Processing

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

scholarly journals Application of high-throughput sequencing (HTS) metabarcoding to diatom biomonitoring: Do DNA extraction methods matter?

2017 ◽  
Vol 36 (1) ◽  
pp. 162-177 ◽  
Author(s):  
Valentin Vasselon ◽  
Isabelle Domaizon ◽  
Frédéric Rimet ◽  
Maria Kahlert ◽  
Agnès Bouchez
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Extraction Methods ◽  
Dna Extraction Methods

scholarly journals Microbial diversity of extreme habitats in human homes

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2376 ◽  
Author(s):  
Amy M. Savage ◽  
Justin Hills ◽  
Katherine Driscoll ◽  
Daniel J. Fergus ◽  
Amy M. Grunden ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Extreme Environments ◽  
Extreme Temperature ◽  
Hot Water ◽  
Extreme Conditions ◽  
Extreme Temperatures ◽  
Environmental Extremes ◽  
Extreme Habitats

High-throughput sequencing techniques have opened up the world of microbial diversity to scientists, and a flurry of studies in the most remote and extreme habitats on earth have begun to elucidate the key roles of microbes in ecosystems with extreme conditions. These same environmental extremes can also be found closer to humans, even in our homes. Here, we used high-throughput sequencing techniques to assess bacterial and archaeal diversity in the extreme environments inside human homes (e.g., dishwashers, hot water heaters, washing machine bleach reservoirs, etc.). We focused on habitats in the home with extreme temperature, pH, and chemical environmental conditions. We found a lower diversity of microbes in these extreme home environments compared to less extreme habitats in the home. However, we were nonetheless able to detect sequences from a relatively diverse array of bacteria and archaea. Habitats with extreme temperatures alone appeared to be able to support a greater diversity of microbes than habitats with extreme pH or extreme chemical environments alone. Microbial diversity was lowest when habitats had both extreme temperature and one of these other extremes. In habitats with both extreme temperatures and extreme pH, taxa with known associations with extreme conditions dominated. Our findings highlight the importance of examining interactive effects of multiple environmental extremes on microbial communities. Inasmuch as taxa from extreme environments can be both beneficial and harmful to humans, our findings also suggest future work to understand both the threats and opportunities posed by the life in these habitats.

scholarly journals A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

2020 ◽  
Author(s):  
Justin P. Shaffer ◽  
Clarisse Marotz ◽  
Pedro Belda-Ferre ◽  
Cameron Martino ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Sequence Data ◽  
Rna Virus ◽  
Microbial Community Composition ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Dna And Rna

AbstractOne goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols.Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX).Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

scholarly journals Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples

PLoS ONE ◽  
2018 ◽  
Vol 13 (5) ◽  
pp. e0197456 ◽  
Author(s):  
Stine H. Kresse ◽  
Heidi M. Namløs ◽  
Susanne Lorenz ◽  
Jeanne-Marie Berner ◽  
Ola Myklebost ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Dna And Rna ◽  
Ffpe Samples ◽  
Rna Extraction Methods

scholarly journals Microbial diversity of extreme habitats in human homes

2016 ◽  
Author(s):  
Amy M. Savage ◽  
Justin Hills ◽  
Katherine Driscoll ◽  
Daniel J Fergus ◽  
Amy M Grunden ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Extreme Environments ◽  
Extreme Temperature ◽  
Hot Water ◽  
Extreme Conditions ◽  
Extreme Temperatures ◽  
Environmental Extremes ◽  
Extreme Habitats

Background: High throughput sequencing techniques have opened up the world of microbial diversity to scientists, and a flurry of studies in the most remote and extreme habitats on earth have begun to elucidate the key roles of microbes in ecosystems with extreme conditions. These same environmental extremes can also be found closer to humans; in fact, they can be found in our homes. Here, we used high throughput sequencing techniques to assess microbial diversity in the extreme environments inside human homes (e.g. dishwashers, hot water heaters, washing machine bleach reservoirs, etc.). We focused on habitats in the home with extreme temperature, pH and chemical environmental conditions. Results: We found that although these habitats supported a lower diversity of microbes than less extreme habitats in the home, there were still diverse microbial assemblages in extreme home environments. Habitats with extreme temperatures alone appeared to be able to support a greater diversity of microbes than habitats with extreme pH or extreme chemical environments alone. Microbial diversity was lowest when habitats had both extreme temperature and one of these other extremes. This interactive effect was strongest when habitats had both extreme temperatures and extreme pH. Under these conditions, taxa with known associations with extreme conditions dominated. Conclusions: Our findings highlight the importance of examining interactive effects of multiple environmental extremes on microbial communities. Inasmuch as taxa from extreme environments can be both pathogens and industrially useful, our findings also suggest future work to understand both the threats and opportunities posed by the life in these habitats.

scholarly journals Towards high–throughput analyses of fecal samples from wildlife

2020 ◽  
pp. 171-183
Author(s):  
C. Sarabia ◽  
I. Salado ◽  
A. Cornellas ◽  
A. Fernández-Gil ◽  
C. Vilà ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Molecular Ecology ◽  
Extraction Methods ◽  
Success Rates ◽  
Sample Collection ◽  
Field Sampling ◽  
Fecal Samples

High–throughput sequencing offers new possibilities in molecular ecology and conservation studies. However, its potential has not yet become fully exploited for noninvasive studies of free–ranging animals, such as those based on feces. High–throughput sequencing allows sequencing of short DNA fragments and could allow simultaneous genotyping of a very large number of samples and markers at a low cost. The application of high throughput genotyping to fecal samples from wildlife has been hindered by several labor–intensive steps. We evaluate alternative protocols which could allow higher throughput for two of these steps: sample collection and DNA extraction. Two different field sampling and seven different DNA extraction methods are tested here on grey wolf (Canis lupus) feces. There was high variation in genotyping success rates. The field sampling method based on surface swabbing performed much worse than the extraction from a fecal fragment. In addition, there is a lot of room for improvement in the DNA extraction step. Optimization of protocols can lead to very much more efficient, cheaper and higher throughput noninvasive monitoring. Selection of appropriate markers is still of paramount importance to increase genotyping success.

Comparing the performance of three ancient DNA extraction methods for high-throughput sequencing

2015 ◽  
Vol 16 (2) ◽  
pp. 459-469 ◽  
Author(s):  
Cristina Gamba ◽  
Kristian Hanghøj ◽  
Charleen Gaunitz ◽  
Ahmed H. Alfarhan ◽  
Saleh A. Alquraishi ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Ancient Dna ◽  
High Throughput Sequencing ◽  
Extraction Methods ◽  
Dna Extraction Methods

scholarly journals Microbial diversity of extreme habitats in human homes

2016 ◽  
Author(s):  
Amy M. Savage ◽  
Justin Hills ◽  
Katherine Driscoll ◽  
Daniel J Fergus ◽  
Amy M Grunden ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Extreme Environments ◽  
Extreme Temperature ◽  
Hot Water ◽  
Extreme Conditions ◽  
Extreme Temperatures ◽  
Environmental Extremes ◽  
Extreme Habitats

High throughput sequencing techniques have opened up the world of microbial diversity to scientists, and a flurry of studies in the most remote and extreme habitats on earth have begun to elucidate the key roles of microbes in ecosystems with extreme conditions. These same environmental extremes can also be found closer to humans, even in our homes. Here, we used high throughput sequencing techniques to assess bacterial and archaeal diversity in the extreme environments inside human homes (e.g., dishwashers, hot water heaters, washing machine bleach reservoirs, etc.). We focused on habitats in the home with extreme temperature, pH and chemical environmental conditions. We found a lower diversity of microbes in these extreme home environments compared to less extreme habitats in the home. However, we were nonetheless able to detect sequences from a relatively diverse array of bacteria and archaea. Habitats with extreme temperatures alone appeared to be able to support a greater diversity of microbes than habitats with extreme pH or extreme chemical environments alone. Microbial diversity was lowest when habitats had both extreme temperature and one of these other extremes. In habitats with both extreme temperatures and extreme pH, taxa with known associations with extreme conditions dominated. Our findings highlight the importance of examining interactive effects of multiple environmental extremes on microbial communities. Inasmuch as taxa from extreme environments can be both beneficial and harmful to humans, our findings also suggest future work to understand both the threats and opportunities posed by the life in these habitats.

Sign in / Sign up

Export Citation Format

Share Document