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

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 Optimisation of DNA extraction from the crustaceanDaphnia

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2004 ◽  
Author(s):  
Camila Gonçalves Athanasio ◽  
James K. Chipman ◽  
Mark R. Viant ◽  
Leda Mirbahai
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Organic Contaminants ◽  
High Throughput Sequencing ◽  
Model Organisms ◽  
High Yield ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Mate Pair

Daphniaare key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA fromDaphniaspp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that ifDaphnia magnaare homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin inDaphnia’scarapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses.

scholarly journals Long-Read Metagenomics to Retrieve High-Quality Metagenome-Assembled Genomes from Canine Feces

2020 ◽  
Author(s):  
Anna Cusco ◽  
Daniel Perez ◽  
Joaquim Viñes ◽  
Olga Francino
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Rrna Genes ◽  
Trna Genes ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Fecal Microbiome ◽  
Long Read ◽  
Gene Similarity

Abstract Background. Metagenomics is a powerful and rapidly developing approach that provides new biological insights into the microbes inhabiting underexplored environments, such as canine fecal microbiome. We investigate long-read metagenomics with Nanopore sequencing to profile the fecal microbiome and to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a healthy dog.Results. More than 99% of total classified reads corresponded to Bacteria. The most abundant phylum was Bacteroidetes (~80% of total reads), followed by Firmicutes, Proteobacteria, and Fusobacteria. Prevotella (>50%) and Bacteroides (>20%) are the more abundant genera, followed by Fusobacterium, Megamonas, Sutterella, and other fecal-related genera, (each representing <5% of the total bacterial composition). We retrieved eight single-contig HQ MAGs and three medium-quality MAGs, after combining several metagenome dataset assemblies. The HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Enterococcus, Blautia, and Catenibacterium genera. Succinivibrio HQ MAG represents a novel candidate bacterial species. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Prevotellamassilia, Phascolarctobacterium, Catenibacterium, and Blautia sp900541345 HQ MAGs improved the contiguity of previously reported genome assemblies in their respective genera, and the number of rRNA genes and tRNA genes. Finally, Enterococcus hirae and Blautia sp003287895 HQ MAGs represented species that already have a complete reference genome. At the technical level, we demonstrated that a high-molecular weight DNA extraction improved the taxonomic classification of the raw unassembled reads, the metagenomics assembly contiguity, and the retrieval of longer and circular contigs, which are potential HQ MAGs. Conclusions. Long-read metagenomics allowed us to recover HQ MAGs from canine feces of a healthy dog. The high-molecular weight DNA extraction to improve contiguity and the correction of the insertions and deletions to reduce frameshift errors ensure the retrieval of complete single-contig HQ MAGs.

scholarly journals Genomic DNA isolation from scrophularieae dried leaves using a simple, high-throughput protocol

2019 ◽  
Vol 48 (4) ◽  
pp. 1231-1235
Author(s):  
Mehrshid Riahi ◽  
Melina Babaei ◽  
Farrokh Ghahremaninejad
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
High Throughput ◽  
Genomic Dna ◽  
Dna Isolation ◽  
High Molecular Weight Dna ◽  
Plant Dna ◽  
Genomic Dna Isolation

This communication described efficient DNA extraction from Scrophularia and Verbascum samples. Modified Murray and Thompson modified Cota-Sànchez method and Bioflux kit methods were applied for the extraction of DNA. Among the different methods, Bioflux kit Plant DNA extraction kit, coupled with some modification was the best for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods.

Next-Generation Sequencing: An Emerging Tool for Drug Designing

2019 ◽  
Vol 25 (31) ◽  
pp. 3350-3357 ◽  
Author(s):  
Pooja Tripathi ◽  
Jyotsna Singh ◽  
Jonathan A. Lal ◽  
Vijay Tripathi
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Large Scale ◽  
Massively Parallel Sequencing ◽  
Genomic Research ◽  
Biological Research ◽  
Next Generation ◽  
Human Welfare ◽  
Drug Designing ◽  
Generation Sequencing

Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.

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