Integrated DNA Extraction Protocol to Avoid PCR-Inhibitors from Fecal and Environmental Samples for Next-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

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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P1.02-059 Evaluation of Plasma DNA Extraction, Droplet PCR and Droplet next Generation Sequencing Methods for Liquid Biopsy Analysis

Journal of Thoracic Oncology ◽

10.1016/j.jtho.2016.11.643 ◽

2017 ◽

Vol 12 (1) ◽

pp. S523-S524

Author(s):

Lina Salleh ◽

Michelle Rozario ◽

Xue Qing Koh ◽

Ross Soo ◽

Richie Soong

Keyword(s):

Next Generation Sequencing ◽

Dna Extraction ◽

Liquid Biopsy ◽

Next Generation ◽

Plasma Dna ◽

Droplet Pcr ◽

Generation Sequencing

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DNA Extraction from Vegetative Tissue for Next-Generation Sequencing

Methods in Molecular Biology - Cereal Genomics ◽

10.1007/978-1-62703-715-0_1 ◽

2013 ◽

pp. 1-5 ◽

Cited By ~ 11

Author(s):

Agnelo Furtado

Keyword(s):

Next Generation Sequencing ◽

Dna Extraction ◽

Next Generation ◽

Vegetative Tissue ◽

Generation Sequencing

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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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Design of genus-specific primer panel for detection and identification of viral DNA in environmental samples using next-generation sequencing

10.18699/bgrssb-2018-026 ◽

2018 ◽

pp. 49-49

Keyword(s):

Next Generation Sequencing ◽

Environmental Samples ◽

Next Generation ◽

Specific Primer ◽

Viral Dna ◽

Detection And Identification ◽

Generation Sequencing

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Comparative analysis of metagenomic DNA extraction methods from gut microbiota of zebrafish (Danio rerio) for downstream next-generation sequencing

Journal of Applied Biology & Biotechnology ◽

10.7324/jabb.2019.70103 ◽

2019 ◽

Vol 7 (1) ◽

pp. 11-15 ◽

Cited By ~ 2

Keyword(s):

Comparative Analysis ◽

Next Generation Sequencing ◽

Gut Microbiota ◽

Dna Extraction ◽

Danio Rerio ◽

Extraction Methods ◽

Next Generation ◽

Metagenomic Dna ◽

Dna Extraction Methods ◽

Generation Sequencing

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A DNA extraction protocol for improved DNA yield from individual mosquitoes

F1000Research ◽

10.12688/f1000research.7413.1 ◽

2015 ◽

Vol 4 ◽

pp. 1314 ◽

Cited By ~ 15

Author(s):

Catelyn C. Nieman ◽

Youki Yamasaki ◽

Travis C. Collier ◽

Yoosook Lee

Keyword(s):

Next Generation Sequencing ◽

Dna Extraction ◽

Whole Genome Amplification ◽

Whole Genome ◽

Single Individual ◽

Dna Amount ◽

Dna Yield ◽

Extraction Protocol ◽

Genome Representation ◽

Generation Sequencing

Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people’s own DNA extraction protocols and not as an advertisement of a commercially available product.

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