Genomic DNA extraction from the pennate diatom Asterionella formosa optimised for next generation sequencing. v1 (protocols.io.jytcpwn)

protocols.io ◽  
2017 ◽  
Author(s):  
Carine Puppo ◽  
Tomas Voisin ◽  
Brigitte Gontero
Keyword(s):  
Next Generation Sequencing ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Next Generation ◽  
Pennate Diatom ◽  
Genomic Dna Extraction ◽  
Asterionella Formosa ◽  
Generation Sequencing

scholarly journals Targeted enrichment of genomic DNA regions for next-generation sequencing

2011 ◽  
Vol 10 (6) ◽  
pp. 374-386 ◽  
Author(s):  
F. Mertes ◽  
A. ElSharawy ◽  
S. Sauer ◽  
J. M. L. M. van Helvoort ◽  
P. J. van der Zaag ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Next Generation ◽  
Generation Sequencing ◽  
Targeted Enrichment

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

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

scholarly journals Co-extraction of genomic DNA & total RNA from recalcitrant woody tissues for next-generation sequencing studies

2018 ◽  
Vol 4 (6) ◽  
pp. FSO309
Author(s):  
Zhen Zeng ◽  
Tommaso Raffaello ◽  
Meng-Xia Liu ◽  
Fred O Asiegbu
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Next Generation ◽  
Total Rna ◽  
Sequencing Studies ◽  
Woody Tissues ◽  
Generation Sequencing

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 Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential

Genes ◽  
2014 ◽  
Vol 5 (4) ◽  
pp. 1115-1131 ◽  
Author(s):  
Yukio Kurihara ◽  
Yuko Makita ◽  
Mika Kawashima ◽  
Hidefumi Hamasaki ◽  
Yoshiharu Yamamoto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Dna Fragments ◽  
Next Generation ◽  
Regulatory Potential ◽  
Generation Sequencing

scholarly journals Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy

2020 ◽  
Vol 8 (1) ◽  
pp. e000299
Author(s):  
Ping Zhang ◽  
Devika Ganesamoorthy ◽  
Son Hoang Nguyen ◽  
Raymond Au ◽  
Lachlan J Coin ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Cost Effective ◽  
Inverse Pcr ◽  
Nanopore Sequencing ◽  
Next Generation ◽  
Short Read ◽  
Vector Integration ◽  
Integration Sites ◽  
Generation Sequencing

BackgroundAnalysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and large batch runs.MethodsWe used nanopore sequencing to analyze the vector integration sites of T cells transduced by the gammaretroviral vector, SFG.iCasp9.2A.ΔCD19. DNA from oligoclonal cell lines and polyclonal clinical samples were restriction enzyme digested with two 6-cutters,NcoIandBspHI; and the flanking genomic DNA amplified by inverse PCR or cassette ligation PCR. Following nested PCR and barcoding, the amplicons were sequenced on the Oxford Nanopore platform. Reads were filtered for quality, trimmed, and aligned. Custom tool was developed to cluster reads and merge overlapping clusters.ResultsBoth inverse PCR and cassette ligation PCR could successfully amplify flanking genomic DNA, with cassette ligation PCR showing less bias. The 4.8 million raw reads were grouped into 12,186 clusters and 6410 clones. The 3′long terminal repeat (LTR)-genome junction could be resolved within a 5-nucleotide span for a majority of clusters and within one nucleotide span for clusters with ≥5 reads. The chromosomal distributions of the insertional sites and their predilection for regions proximate to transcription start sites were consistent with previous reports for gammaretroviral vector integrants as analyzed by short-read next-generation sequencing.ConclusionOur study shows that it is feasible to use nanopore sequencing to map polyclonal vector integration sites. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis. Further refinement is required to reduce amplification bias and improve single nucleotide resolution.

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