DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

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Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

10.7287/peerj.preprints.3073v1 ◽

2017 ◽

Author(s):

Taha Soliman ◽

Sung-Yin Yang ◽

Tomoko Yamazaki ◽

Holger Jenke-Kodama

Keyword(s):

Next Generation Sequencing ◽

Microbial Communities ◽

Dna Extraction ◽

Dna Isolation ◽

Soil Microbial Communities ◽

Next Generation ◽

Microbial Composition ◽

Okinawa Island ◽

The Impact ◽

Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

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Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

PeerJ ◽

10.7717/peerj.4178 ◽

2017 ◽

Vol 5 ◽

pp. e4178 ◽

Cited By ~ 17

Author(s):

Taha Soliman ◽

Sung-Yin Yang ◽

Tomoko Yamazaki ◽

Holger Jenke-Kodama

Keyword(s):

Next Generation Sequencing ◽

Microbial Communities ◽

Dna Extraction ◽

Dna Isolation ◽

Soil Microbial Communities ◽

Next Generation ◽

Microbial Composition ◽

Okinawa Island ◽

The Impact ◽

Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil®DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin®Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA;P < 0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

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DNA extraction and host depletion methods significantly impact and potentially bias bacterial detection in a biological fluid

10.1101/2020.08.21.262337 ◽

2020 ◽

Author(s):

Erika Ganda ◽

Kristen L. Beck ◽

Niina Haiminen ◽

Ban Kawas ◽

Brittany Cronk ◽

...

Keyword(s):

Food Safety ◽

Nucleic Acid ◽

Nucleic Acids ◽

Biological Fluid ◽

Genetic Material ◽

Bovine Milk ◽

Extraction Methods ◽

Acid Extraction ◽

Primary Material ◽

The Impact

ABSTRACTAbstractUntargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis could reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering primary material, laboratory methods, data analysis, and interpretation.The focus of this study is in gathering the primary material, in this case, DNA. We used bovine milk as a model to 1) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk; 2) evaluate host DNA depletion methods for use with milk, and 3) develop and evaluate a selective lysis-PMA based protocol for host DNA depletion in milk.Our results suggest that magnetic-based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety where the detection of Gram-negative pathogens and indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host.ImportanceTracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of what the relative composition looks like. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around one thousand times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods in the ability of detecting specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.

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Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

Sensors ◽

10.3390/s21020602 ◽

2021 ◽

Vol 21 (2) ◽

pp. 602

Author(s):

Sandra Leonardo ◽

Anna Toldrà ◽

Mònica Campàs

Keyword(s):

Food Safety ◽

Environmental Monitoring ◽

Rolling Circle Amplification ◽

Dna Amplification ◽

Isothermal Amplification ◽

In Situ Monitoring ◽

Strand Displacement ◽

Strand Displacement Amplification ◽

Bacterial Detection ◽

Efficient Detection

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

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Method for rapid DNA extraction from bacterial communities of different soils

Microbiology ◽

10.1134/s0026261706010188 ◽

2006 ◽

Vol 75 (1) ◽

pp. 105-111 ◽

Cited By ~ 4

Author(s):

E. V. Zaporozhenko ◽

N. V. Slobodova ◽

E. S. Boulygina ◽

I. K. Kravchenko ◽

B. B. Kuznetsov

Keyword(s):

Dna Extraction ◽

Bacterial Communities ◽

Different Soils

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Liquid Biopsy in Gastrointestinal Cancers

Diagnostics ◽

10.3390/diagnostics8040075 ◽

2018 ◽

Vol 8 (4) ◽

pp. 75 ◽

Cited By ~ 3

Author(s):

Aman Saini ◽

Yash Pershad ◽

Hassan Albadawi ◽

Malia Kuo ◽

Sadeer Alzubaidi ◽

...

Keyword(s):

Liquid Biopsy ◽

Disease Surveillance ◽

Biological Fluid ◽

Genetic Material ◽

Circulating Tumor Dna ◽

Cancer Diagnostics ◽

Gastrointestinal Cancers ◽

New Paradigm ◽

Liquid Biopsies ◽

Biopsy Material

Liquid biopsy is the sampling of any biological fluid in an effort to enrich and analyze a tumor’s genetic material. Peripheral blood remains the most studied liquid biopsy material, with circulating tumor cells (CTC’s) and circulating tumor DNA (ctDNA) allowing the examination and longitudinal monitoring of a tumors genetic landscape. With applications in cancer screening, prognostic stratification, therapy selection and disease surveillance, liquid biopsy represents an exciting new paradigm in the field of cancer diagnostics and offers a less invasive and more comprehensive alternative to conventional tissue biopsy. Here, we examine liquid biopsies in gastrointestinal cancers, specifically colorectal, gastric, and pancreatic cancers, with an emphasis on applications in diagnostics, prognostics and therapeutics.

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Comparative Analysis of the Gut Microbiota of Adult Mosquitoes From Eight Locations in Hainan, China

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.596750 ◽

2020 ◽

Vol 10 ◽

Author(s):

Xun Kang ◽

Yanhong Wang ◽

Siping Li ◽

Xiaomei Sun ◽

Xiangyang Lu ◽

...

Keyword(s):

Gut Microbiota ◽

Disease Control ◽

Bacterial Communities ◽

Mosquito Species ◽

Microbial Community Composition ◽

Variable Region ◽

Rrna Gene ◽

Microbial Composition ◽

Quality Filtering ◽

And Function

The midgut microbial community composition, structure, and function of field-collected mosquitoes may provide a way to exploit microbial function for mosquito-borne disease control. However, it is unclear how adult mosquitoes acquire their microbiome, how the microbiome affects life history traits and how the microbiome influences community structure. We analyzed the composition of 501 midgut bacterial communities from field-collected adult female mosquitoes, including Aedes albopictus, Aedes galloisi, Culex pallidothorax, Culex pipiens, Culex gelidus, and Armigeres subalbatus, across eight habitats using the HiSeq 4000 system and the V3−V4 hyper-variable region of 16S rRNA gene. After quality filtering and rarefaction, a total of 1421 operational taxonomic units, belonging to 29 phyla, 44 families, and 43 genera were identified. Proteobacteria (75.67%) were the most common phylum, followed by Firmicutes (10.38%), Bacteroidetes (6.87%), Thermi (4.60%), and Actinobacteria (1.58%). The genera Rickettsiaceae (33.00%), Enterobacteriaceae (20.27%), Enterococcaceae (7.49%), Aeromonadaceae (7.00%), Thermaceae (4.52%), and Moraxellaceae (4.31%) were dominant in the samples analyzed and accounted for 76.59% of the total genera. We characterized the midgut bacterial communities of six mosquito species in Hainan province, China. The gut bacterial communities were different in composition and abundance, among locations, for all mosquito species. There were significant differences in the gut microbial composition between some species and substantial variation in the gut microbiota between individuals of the same mosquito species. There was a marked variation in different mosquito gut microbiota within the same location. These results might be useful in the identification of microbial communities that could be exploited for disease control.

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