scholarly journals SILEX: A fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

2020 ◽  
Author(s):  
santiago vilanova ◽  
David Alonso ◽  
Pietro Gramazio ◽  
Mariola Plazas ◽  
Edgar Garcia Fortea ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Next Generation Sequencing ◽  
Dna Extraction ◽  
High Throughput ◽  
Extraction Method ◽  
High Quality ◽  
Extraction Protocol ◽  
Dna Extraction Method ◽  
Sequencing Platforms ◽  
Generation Sequencing

Abstract Background The use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomic DNA (gDNA) in sufficient concentrations is often a main limitation, especially for third-generation sequencing platforms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly and frequently give either low yield or low-quality DNA, inappropriate for next generation sequencing (NGS) platforms. Here, we describe a fast and inexpensive DNA extraction method (SILEX) applicable to a wide range of plant species and tissues. Results SILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silica matrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitory compounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kit in a variety of species, including recalcitrant ones, from different families. In comparison with the other methods, SILEX yielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 min by one person at a cost of 0.12 €/sample of reagents and consumables. Hundreds of tomato gDNA samples obtained with either SILEX or the commercial kit were successfully genotyped with Single Primer Enrichment Technology (SPET) with the Illumina HiSeq 2500 platform. Furthermore, DNA extracted from Solanum elaeagnifolium using this protocol was assessed by Pulsed-field gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencing platforms that required high-molecular-weight DNA such as Nanopore or PacBio. Conclusions A high-throughput, fast and inexpensive DNA extraction protocol was developed and validated for a wide variety of plants and tissues. SILEX offers an easy, scalable, efficient and inexpensive way to extract DNA for various next-generation sequencing applications including SPET and Nanopore among others.

scholarly journals SILEX: A fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

2020 ◽  
Author(s):  
santiago vilanova ◽  
David Alonso ◽  
Pietro Gramazio ◽  
Mariola Plazas ◽  
Paola Ferrante ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Next Generation Sequencing ◽  
Dna Extraction ◽  
High Throughput ◽  
Extraction Method ◽  
High Quality ◽  
Extraction Protocol ◽  
Dna Extraction Method ◽  
Sequencing Platforms ◽  
Generation Sequencing

Abstract Background: The use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomic DNA (gDNA) in sufficient concentrations is often a main limitation, especially for third-generation sequencing platforms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly and frequently give either low yield or low-quality DNA, inappropriate for next generation sequencing (NGS) platforms. Here, we describe a fast and inexpensive DNA extraction method (SILEX) applicable to a wide range of plant species and tissues. Results: SILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silica matrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitory compounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kit in a variety of species, including recalcitrant ones, from different families. In comparison with the other methods, SILEX yielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 min by one person at a cost of 0.12 €/sample of reagents and consumables. Hundreds of tomato gDNA samples obtained with either SILEX or the commercial kit were successfully genotyped with Single Primer Enrichment Technology (SPET) with the Illumina HiSeq 2500 platform. Furthermore, DNA extracted from Solanum elaeagnifolium using this protocol was assessed by Pulsed-field gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencing platforms that required high-molecular-weight DNA such as Nanopore or PacBio. Conclusions: A high-throughput, fast and inexpensive DNA extraction protocol was developed and validated for a wide variety of plants and tissues. SILEX offers an easy, scalable, efficient and inexpensive way to extract DNA for various next-generation sequencing applications including SPET and Nanopore among others.

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.

scholarly journals SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Santiago Vilanova ◽  
David Alonso ◽  
Pietro Gramazio ◽  
Mariola Plazas ◽  
Edgar García-Fortea ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Plant Species ◽  
Extraction Method ◽  
High Quality ◽  
Dna Extraction Method ◽  
Sequencing Platforms

scholarly journals Cost-Effective and Scalable DNA Extraction Method from Dried Blood Spots

2013 ◽  
Vol 59 (7) ◽  
pp. 1045-1051 ◽  
Author(s):  
Carlos A Saavedra-Matiz ◽  
Jason T Isabelle ◽  
Chad K Biski ◽  
Salvatore J Duva ◽  
Melissa L Sweeney ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Extraction Method ◽  
Low Cost ◽  
Melting Curve Analysis ◽  
Molecular Testing ◽  
High Quality ◽  
Congenital Diseases ◽  
Cell Counts ◽  
Dna Extraction Method

BACKGROUND Dried blood spot (DBS) samples have been widely used in newborn screening (NBS) for the early identification of disease to facilitate the presymptomatic treatment of congenital diseases in newborns. As molecular genetics knowledge and technology progresses, there is an increased demand on NBS programs for molecular testing and a need to establish reliable, low-cost methods to perform those analyses. Here we report a flexible, cost-efficient, high-throughput DNA extraction method from DBS adaptable to small- and large-scale screening settings. METHODS Genomic DNA (g.DNA) was extracted from single 3-mm diameter DBS by the sequential use of red cell lysis, detergent-alkaline, and acid-neutralizing buffers routinely used in whole blood and plant tissue DNA extractions. We performed PCR amplification of several genomic regions using standard PCR conditions and detection methods (agarose gel, melting-curve analysis, TaqMan-based assays). Amplicons were confirmed by BigDye® Terminator cycle sequencing and compared with reference sequences. RESULTS High-quality g.DNA was extracted from hundreds of DBS, as proven by mutation detection of several human genes on multiple platforms. Manual and automated extraction protocols were validated. Quantification of g.DNA by Oligreen® fluorescent nucleic acid stain demonstrated a normal population distribution closely corresponding with white blood cell counts detected in newborn populations. CONCLUSIONS High-quality, amplifiable g.DNA is extractable from DBSs. Our method is adaptable, reliable, and scalable to low- and high-throughput NBS at low cost ($0.10/sample). This method is routinely used for molecular testing in the New York State NBS program.

scholarly journals DNA Extraction from Bronchial Aspirates for Molecular Cytology: Which Method to Take?

2003 ◽  
Vol 25 (2) ◽  
pp. 83-88 ◽  
Author(s):  
Hans Jürgen Grote ◽  
Viola Schmiemann ◽  
Mario Sarbia ◽  
Alfred Böcking
Keyword(s):  
Success Rate ◽  
Dna Extraction ◽  
Extraction Method ◽  
Globin Gene ◽  
Extraction Methods ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Dna Yield ◽  
Extraction Protocol ◽  
Dna Extraction Method

Objective: To date, there are only few systematic reports on the quality of DNA extracted from routine diagnostic cytologic specimens. It was the aim of the present study to evaluate the ability of 50% ethanol/2% carbowax (Saccomanno fixative) to preserve bronchial secretions with high quality genomic DNA as well as to compare different DNA extraction methods.Methods: DNA was extracted from 45 bronchial aspirates by four different extraction protocols. Beside DNA yield, DNA quality with regard to purity, integrity, and PCR success rate were investigated.Results: No fragmentation of sample DNA due to the fixative was detected. It was preserved as high molecular weight DNA. DNA yield, purity, and integrity were dependent on the DNA extraction method to some extend. Irrespective of the DNA extraction method the PCR success rate for amplification of β‐globin gene fragments (268, 536, and 989 bp) was 100%. Conclusions: A fixative containing 50% ethanol/2% carbowax preserves high quality DNA which is well suited for PCR‐based assays regardless of the extraction protocol used. The selection of the DNA extraction protocol has to be adjusted to the circumstances of application.

scholarly journals A new, harmless, high-throughput endosperm-based DNA extraction method for wheat

2019 ◽  
Vol 49 (9) ◽  
Author(s):  
Zhihui Ma ◽  
Yuquan Wang ◽  
Wenhui Wei ◽  
Zhengang Ru
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Extraction Method ◽  
Seedling Emergence ◽  
Control Group ◽  
Suitable Alternative ◽  
Dna Extraction Method ◽  
High Throughput Method ◽  
Young Leaves ◽  
Non Destructive

ABSTRACT: In this study, a non-destructive, high-throughput, endosperm-based DNA extraction method was developed. To verify the non-destructive nature of this method, a germination test was performed on 288 seeds after sampling their endosperm, which gave a seedling emergence rate that was higher (97.6%) than that of the control group (92%). To confirm the feasibility of the new method, DNA was extracted from plants of a BC1F2 population by two different methods, namely, from endosperm using our rapid, high-throughput method (ER-DNA) and from young leaves emerging from the same sampled seed using the CTAB method (LC-DNA). The ER-DNA was undetectable by agarose gel electrophoresis, but was found to be an adequate replacement for LC-DNA for the amplification and detection of simple sequence repeats (SSRs). Further analysis revealed that ER-DNA was generally suitable for the generation of specific 500-750-bp fragments, but not for the amplification of 1,000-2,000-bp fragments. Our rapid, high-throughput method therefore has no deleterious effects on wheat seeds and yields DNA for SSR genotyping that is a suitable alternative to traditionally obtained DNA.

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