Extraction Of High 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.

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.

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.

High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.

Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30-50 kb, with reads exceeding 200 kb and outputs ranging from 15-30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria samples.

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.

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.

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

Optimization of DNA extraction from seeds and leaf tissues of Chrysanthemum (Chrysanthemum indicum) for polymerase chain reaction.

Chrysanthemums constitute approximately 30 species of perennial flowering plants, belonging to the family Asteraceae, native to Asia and Northeastern Europe. Chrysanthemum is a natural cosmetic additive extracted from Chinese herb by modern biochemical technology. It has the properties of anti-bacterial, anti-viral, reducing (detoxification) and anti-inflammation. It possesses antioxidant characteristics, which could assist in minimizing free-radical induced damage. Therefore, it is widely used in skin and hair care products. Chemical composition of this herbal remedy includes kikkanols, sesquiterpenes, flavonoids, various essential oils containing camphor, cineole, sabinol, borneole and other elements that interfere with DNA, causing erroneous or no PCR products. In the present study, testing and modification of various standard protocols for isolation of high-quality DNA from leaf tissues and seeds of C. indicum was done. It was observed that the DNA obtained from seeds and leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. Also, DNA could be extracted from leaf tissues without using liquid nitrogen. Quality of DNA extracted from seeds was much better as compared to that extracted from leaf tissues. The extracted DNA was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extraction of high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils.

Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high-salt protein fraction for quantitative proteome analysis

Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high-molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high-salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2-D DIGE, MS and Western blotting.

High molecular weight DNA extraction from Arabidopsis.

High molecular weight DNA extraction from Arabidopsis.