Extraction Of Molecular Weight DNA Research Articles

High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing

Abstract. Jasim JHM, Othman AS, Nordin FA, Talkah NSM. 2024. High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing. Biodiversitas 25: 654-663. Advancements in genomic research have spurred a growing interest in extracting high-quality genomic DNA, particularly from intricate plant species like the Yellow Spathoglottis Blume Complex. The preparation of genomic DNA high-quality from a plant sample is a crucial step for genomic and genetic analysis studies. A variety of genomic DNA extraction methods such as the traditional Hexadecyltrimethylammonium Bromide (CTAB) method and commercially available kits have been reported. Even so, they are either low purity and yield or costly. We develop a good-quality genomic DNA extraction method from fresh and dried leaves of the Yellow Spathoglottis Blume complex in Peninsular Malaysia, specifically tailored for Next-Generation Sequencing (NGS) applications. Three DNA extraction methods were compared traditional CTAB, modified CTAB, and the DNeasy Qiagen plant mini kit methods. The yield and quality of extracted DNA were assessed by PCR amplification using nuclear ETS and ITS genes performed to evaluate the success of DNA extraction. Additionally, the suitability of the modified CTAB method for NGS library preparation and sequencing was tested. It demonstrated better PCR amplification results, yielding high-concentration bands for both ETS and ITS primers. Furthermore, the modified CTAB method proved suitable for high molecular weight DNA extraction, without contamination. The extracted DNA from Spathoglottis aurea exhibited high molecular weight and passed the quality control assessment for NGS library preparation. Sequencing results demonstrated an average quality score of 36.0, with an accurate base call rate of approximately 99.9%. The developed method enables reliable genomic analysis and sequencing of the Yellow Spathoglottis Blume complex, contributing to further research and understanding of this species group.

Extracting high-molecular weight DNA from cyanobacteria using Promega's WizardⓇ HMW DNA extraction kit with a modified protocol, METIS

Extraction of high molecular weight (HMW) DNA for long read sequencing with little to no fragmentation and high purity is difficult to acquire from cyanobacterial species. Here we describe a modified method of extraction using Promega's WizardⓇ HMW DNA Extraction Kit to acquire high molecular weight DNA from two cyanobacterial species. The protocol used in the kit is the “3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria” protocol. During a key step in the protocol, we propose that the lingering remnants of the cellular debris such as the mucilage layer of the cyanobacterial species is removed, preventing it from sticking to the DNA pellet produced. This customized modification is done between steps 11 and 12 and called METIS (maximizing extraction, transfer isopropanol step). This step drastically reduces the remaining mucilage layer, which if kept will stick to the DNA and make the DNA unsuitable for sensitive downstream next generation sequencing, like PacBio Sequencing. This protocol has been used to assemble two genomes from cyanobacteria (Synechococcus sp. and Microcystis aeruginosa) and one from a gram-negative bacterium, Lacibacter. It also allows for HMW DNA to be rapidly extracted without the use of toxic chemicals such as phenol and without extra reagents to be purchased.•Maximizing extraction, transfer isopropanol step (METIS) is the key modification during the step of DNA unraveling•METIS reduces leftover remnants of the mucilage layer in the extraction•High molecular weight DNA is produced with little to no fragmentation, and both a high purity and concentration

A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants

BackgroundPacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing.ResultsWe developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively.ConclusionsDNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.

Tissue Cultivation, Preparation, and Extraction of High Molecular Weight DNA for Single-Molecule Genome Sequencing of Plant-Associated Fungi.

Extraction of high-quality, high molecular weight DNA is a critical step for sequencing an organism's genome. For fungi, DNA extraction is often complicated by co-precipitation of secondary metabolites, the most destructive being polysaccharides, polyphenols, and melanin. Different DNA extraction protocols and clean-up methods have been developed to address challenging materials and contaminants; however, the method of fungal cultivation and tissue preparation also plays a critical role to limit the production of inhibitory compounds prior to extraction. Here, we provide protocols and guidelines for (i) fungal tissue cultivation and processing with solid media containing a cellophane overlay or in liquid media, (ii) DNA extraction with customized recommendations for taxonomically and ecologically diverse plant-associated fungi, and (iii) assessing DNA quantity and quality for downstream genome sequencing with single-molecule technology such as PacBio.

Genome Assembly of the Polyclad Flatworm Prostheceraeus crozieri.

Polyclad flatworms are widely thought to be one of the least derived of the flatworm classes and, as such, are well placed to investigate evolutionary and developmental features such as spiral cleavage and larval diversification lost in other platyhelminths. Prostheceraeus crozieri, (formerly Maritigrella crozieri), is an emerging model polyclad flatworm that already has some useful transcriptome data but, to date, no sequenced genome. We have used high molecular weight DNA extraction and long-read PacBio sequencing to assemble the highly repetitive (67.9%) P. crozieri genome (2.07 Gb). We have annotated 43,325 genes, with 89.7% BUSCO completeness. Perhaps reflecting its large genome, introns were considerably larger than other free-living flatworms, but evidence of abundant transposable elements suggests genome expansion has been principally via transposable elements activity. This genome resource will be of great use for future developmental and phylogenomic research.

Evaluation of high molecular weight DNA extraction methods for long-read sequencing of Shiga toxin-producing Escherichia coli

Next generation sequencing has become essential for pathogen characterization and typing. The most popular second generation sequencing technique produces data of high quality with very low error rates and high depths. One major drawback of this technique is the short reads. Indeed, short-read sequencing data of Shiga toxin-producing Escherichia coli (STEC) are difficult to assemble because of the presence of numerous mobile genetic elements (MGEs), which contain repeated elements. The resulting draft assemblies are often highly fragmented, which results in a loss of information, especially concerning MGEs or large structural variations. The use of long-read sequencing can circumvent these problems and produce complete or nearly complete genomes. The ONT MinION, for its small size and minimal investment requirements, is particularly popular. The ultra-long reads generated with the MinION can easily span prophages and repeat regions. In order to take full advantage of this technology it requires High Molecular Weight (HMW) DNA of high quality in high quantity. In this study, we have tested three different extraction methods: bead-based, solid-phase and salting-out, and evaluated their impact on STEC DNA yield, quality and integrity as well as performance in MinION long-read sequencing. Both the bead-based and salting-out methods allowed the recovery of large quantities of HMW STEC DNA suitable for MinION library preparation. The DNA extracted using the salting-out method consistently produced longer reads in the subsequent MinION runs, compared with the bead-based methods. While both methods performed similarly in subsequent STEC genome assembly, DNA extraction based on salting-out appeared to be the overall best method to produce high quantity of pure HMW STEC DNA for MinION sequencing.

High molecular weight DNA extraction methods lead to high quality filamentous ascomycete fungal genome assemblies using Oxford Nanopore sequencing.

During the last two decades, whole-genome sequencing has revolutionized genetic research in all kingdoms, including fungi. More than 1000 fungal genomes have been submitted to sequence databases, mostly obtained through second generation short-read DNA sequencing. As a result, highly fragmented genome drafts have typically been obtained. However, with the emergence of third generation long-read DNA sequencing, the assembly challenge can be overcome and highly contiguous assemblies obtained. Such attractive results, however, are extremely dependent on the ability to extract highly purified high molecular weight (HMW) DNA. Extraction of such DNA is currently a significant challenge for all species with cell walls, not least fungi. In this study, four isolates of filamentous ascomycetes (Apiospora pterospermum, Aspergillus sp. (subgen. Cremei), Aspergillus westerdijkiae, and Penicillium aurantiogriseum) were used to develop extraction and purification methods that result in HMW DNA suitable for third generation sequencing. We have tested and propose two straightforward extraction methods based on treatment with either a commercial kit or traditional phenol-chloroform extraction both in combination with a single commercial purification method that result in high quality HMW DNA from filamentous ascomycetes. Our results demonstrated that using these DNA extraction methods and coverage, above 75 x of our haploid filamentous ascomycete fungal genomes result in complete and contiguous assemblies.

Methods to Improve Molecular Diagnosis in Genomic Cold Cases in Pediatric Neurology.

During the last decade, genetic testing has emerged as an important etiological diagnostic tool for Mendelian diseases, including pediatric neurological conditions. A genetic diagnosis has a considerable impact on disease management and treatment; however, many cases remain undiagnosed after applying standard diagnostic sequencing techniques. This review discusses various methods to improve the molecular diagnostic rates in these genomic cold cases. We discuss extended analysis methods to consider, non-Mendelian inheritance models, mosaicism, dual/multiple diagnoses, periodic re-analysis, artificial intelligence tools, and deep phenotyping, in addition to integrating various omics methods to improve variant prioritization. Last, novel genomic technologies, including long-read sequencing, artificial long-read sequencing, and optical genome mapping are discussed. In conclusion, a more comprehensive molecular analysis and a timely re-analysis of unsolved cases are imperative to improve diagnostic rates. In addition, our current understanding of the human genome is still limited due to restrictions in technologies. Novel technologies are now available that improve upon some of these limitations and can capture all human genomic variation more accurately. Last, we recommend a more routine implementation of high molecular weight DNA extraction methods that is coherent with the ability to use and/or optimally benefit from these novel genomic methods.

High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes.

By offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long‐read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long‐read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short‐read sequencing. The challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long‐read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol–chloroform (PC) extraction and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions, as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read‐size distribution, assembly performance and the number of circularized elements found in sequencing results suggest that column‐based kits with enzyme supplementation, rather than PC methods, may be more appropriate for long‐read sequencing of metagenomes.

An optimized method for high-quality DNA extraction medicinal fungi Mycoleptodonoides aitchisonii for whole genome sequencing

The extraction of pure high molecular weight DNA and collecting genomic DNA from fungi is difficult. This is because filamentous fungi has a sturdy cell wall, high protein and polysaccharide levels resistant to the usual DNA extraction procedures. A low-cost and reliable DNA extraction method was designed from Mycoleptodonoides. aitchisonii fit for whole-genome sequencing for identification and mapping of the A mating-type gene. Mycoleptodonoides. aitchisonii belongs to the Climacodontaceae family has pharmaceutical activities. In the present study, the mycelia of M. aitchisonii, which grew from the different concentrations of malt extract and minimal liquid media, have been compared systematically to determine the DNA extraction procedure resulted in DNA concentration with excellent purity and quality. The best protocol that resulted in good quality DNA was further validated using polymerase chain reaction amplification with a specific primer to amplify the homeodomain protein (Mahd2-18) gene that encodes a transcription factor. The method proposed DNA extraction using CTAB (Cetyl Trimethyl Ammonium Bromide) method and purify by commercial kit from mycelium grown in the minimal liquid media give the best result with the high concentration of DNA for whole-genome sequencing by Next Generation Sequencing and other applications.

Isolation of High Molecular Weight DNA from the Model Beetle Tribolium for Nanopore Sequencing.

The long-read Nanopore sequencing has been recently applied for assembly of complex genomes and analysis of linear genome organization. The most critical factor for successful long-read sequencing is extraction of high molecular weight (HMW) DNA of sufficient purity and quantity. The challenges associated with input DNA quality are further amplified when working with extremely small insects with hard exoskeletons. Here, we optimized the isolation of HMW DNA from the model beetle Tribolium and tested for use in Nanopore sequencing. We succeeded in overcoming all the difficulties in HMW handling and library preparation that were encountered when using published protocols and commercial kits. Isolation of nuclei and subsequent purification of DNA on an anion-exchange chromatography column resulted in genomic HMW DNA that was efficiently relaxed, of optimal quality and in sufficient quantity for Nanopore MinION sequencing. DNA shearing increased average N50 read values up to 26 kb and allowed us to use a single flow cell in multiple library loads for a total output of more than 13 Gb. Although our focus was on T. castaneum and closely related species, we expect that this protocol, with appropriate modifications, could be extended to other insects, particularly beetles.

Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform.

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

A simple method for high molecular-weight genomic DNA extraction suitable for long-read sequencing from spores of an obligate biotroph oomycete

Long-read sequencing technologies are having a major impact on our approaches to studying non-model organisms and microbial communities. By significantly reducing the cost and facilitating the genome assembly pipelines, any laboratory can now develop its own genomics program regardless of the complexity of the genome studied. The most crucial current challenge is to develop efficient protocols for extracting genomic DNA (gDNA) with high quality and integrity adapted to the organism of interest. This can be particularly complex for obligate pathogens that must maintain intimate interactions inside infected host tissues. Here we propose a simple and cost-effective method for high molecular weight gDNA extraction from spores of Plasmopara halstedii, an obligate biotroph oomycete pathogen responsible for downy mildew in sunflower. We optimized the yield, the quality and the integrity of the extracted gDNA by fine-tuning three critical parameters, the grinding, the lysis temperature and the lysis duration. We obtained gDNA with a fragment size distribution reaching a peak ranging from 79 to 145 kb. More than half of the extracted gDNA consisted of DNA fragments larger than 42 kb, with 23% of fragments larger than 100 kb. We then demonstrated the relevance of this protocol for long-read sequencing using PacBio RSII technology. With this protocol, we were able to obtain a mean read length of 9.3 kb, a max read length of 71 kb and an N50 of 13.3 kb. The development of such DNA extraction protocols is an essential prerequisite for fully exploiting technologies requiring high molecular weight gDNA (e.g. long-read sequencing or optical mapping). These technological advances will help generate data to answer questions such as the role of newly duplicated gene clusters, repeated regions, genomic structural variations or to define number of chromosomes that still remains undefined in many species of pathogenic fungi and oomycetes.

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

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.

Ultra-long Read Sequencing for Whole Genomic DNA Analysis.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 µg of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Microfluidic long DNA sample preparation from cells.

A number of outstanding problems in genomics, such as identifying structural variations and sequencing through centromeres and telomeres, stand poised to benefit tremendously from emerging long-read genomics technologies such as nanopore sequencing and genome mapping in nanochannels. However, optimal application of these new genomics technologies requires facile methods for extracting long DNA from cells. These sample preparation tools should be amenable to automation and minimize fragmentation of the long DNA molecules by shear. We present one such approach in a poly(dimethylsiloxane) device, where gel-based high molecular weight DNA extraction and continuous flow purification in a 3D cell culture-inspired geometry is followed by electrophoretic extraction of the long DNA from the miniaturized gel. Molecular combing reveals that the device produces molecules that are typically in excess of 100 kilobase pairs in size, with the longest molecule extending up to 4 megabase pairs. The microfluidic format reduces the standard day-long and labor-intensive DNA extraction process to 4 hours, making it a promising prototype platform for routine long DNA sample preparation.

Comparison of 4 commercial kits for the extraction of circulating DNA from plasma

The utility of circulating DNA as a source of clinical biomarkers in blood is limited by its low concentration and small fragment size. Effective purification methods can maximize circulating DNA yield and contribute to the success of downstream protocols. We describe the evaluation of 4 commercial DNA purification kits-QIAamp Circulating Nucleic Acids kit, QIAamp DNA Blood Mini kit, QIAamp Ultrasens Virus kit and the QIASymphony DSP Virus kit-for the extraction of high and low molecular weight DNA from blood plasma. Using qPCR to quantitate endogenous Alu sequences, as well as spiked exogenous high and low molecular weight zebrafish DNA, we found that the Circulating Nucleic Acids kit and the DSP kit were both efficient at purifying DNA from plasma regardless of fragment size, whereas the DNA Blood Mini kit was only able to effectively extract high molecular weight DNA. The Ultrasens Virus Kit produced the lowest yields for both large and small fragments. The use of carrier RNA with the Circulating Nucleic Acids and the DSP kits improved yields. Appropriate choice of kit can be an important factor in determining experiment outcome.

Root Gall Formation, Resting Spore Isolation and High Molecular Weight DNA Extraction of Plasmodiophora brassicae

Root Gall Formation, Resting Spore Isolation and High Molecular Weight DNA Extraction of Plasmodiophora brassicae

Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing.

Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.

Optimisation of DNA extraction from the crustacean Daphnia.

Daphnia are 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 from Daphnia spp. 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 if Daphnia magna are 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 in Daphnia’s carapace 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.