A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

10.3791/62354 ◽  
2021 ◽  
Author(s):  
Tse-Yu Chen ◽  
Adam E. Vorsino ◽  
Kyle J. Kosinski ◽  
Ana L. Romero-Weaver ◽  
Eva A. Buckner ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Dna Extraction ◽  
Magnetic Bead ◽  
Next Generation ◽  
Extraction Protocol ◽  
Generation Sequencing

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

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.

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

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.

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

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4178 ◽  
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.

scholarly journals A DNA extraction protocol for improved DNA yield from individual mosquitoes

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1314 ◽  
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.

scholarly journals Standardization of DNA extraction from sand flies: Application to genotyping by next generation sequencing

2017 ◽  
Vol 177 ◽  
pp. 66-72 ◽  
Author(s):  
Aline Etelvina Casaril ◽  
Liliane Prado de Oliveira ◽  
Diego Peres Alonso ◽  
Everton Falcão de Oliveira ◽  
Suellem Petilim Gomes Barrios ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Dna Extraction ◽  
Sand Flies ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Extraction of high molecular weight abaca DNA suitable for next-generation sequencing

2020 ◽  
Author(s):  
Rhosener Bhea Lu Koh ◽  
Cris Francis Cortez Barbosa ◽  
Vermando Masinsin Aquino ◽  
Leny Calano Galvez
Keyword(s):  
Molecular Weight ◽  
Next Generation Sequencing ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
High Throughput ◽  
Next Generation ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Ctab Method ◽  
Generation Sequencing

Abstract Background The abaca (Musa textilis Née) is a fiber crop native to the Philippines with high economic value because of its fiber - the Manila hemp, known to be the strongest of all the natural fibers. DNA extraction in abaca is difficult due to its fibrous nature, high cellulose content and polyphenol compounds. Thus an optimized DNA extraction method is required for extracting high quality abaca DNA for next-generation sequencing applications. Results In this study, we have compared five different methods for the extraction of high molecular weight DNA from abaca leaves. The methods are the traditional CTAB method (Protocol 1), the CTAB with PVP method (Protocol 2), the CTAB with 0.3% β-mercaptoethanol method (Protocol 3), SDS-method (Protocol 4) and CTAB with Triton X-100 and PVP method (Protocol 5). Out of the five methods tested, traditional CTAB-method (Protocol 1), CTAB with 0.3% β-mercaptoethanol method (Protocol 3) and SDS-method (Protocol 4) have shown to be the most consistent in giving high molecular weight DNA with good yield and purity based on A260/A280 and A260/A230 absorption values. TissueLyserII was also utilized for homogenization for the three extraction protocols for applications in high-throughput DNA extraction. DNA from two abaca varieties were extracted using the CTAB with 0.3% β-mercaptoethanol method (Protocol 3) and were sent for NGS based on Illumina HiSeq platform having both passed the quality control for library preparation. Conclusion The CTAB with 0.3% β-mercaptoethanol method (Protocol 3) was found to be the simplest and most consistent method for extracting average yield DNA with high quality for NGS applications. The SDS-method (Protocol 4) was determined to have the shortest processing time and together with TissueLyserII is the most appropriate method for high-throughput extraction of abaca samples which will be useful for genotyping-by-sequencing (GBS) studies.

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