High-quality Genomic DNA Research Articles

Extraction of High-quality Genomic DNA Using Species Tailored Protocol for Prosopis cineraria (L.) Druce (Khejri)

To effectively conserve genetic diversity in forestry species, molecular methods are essential. These methods necessitate high-quality genomic DNA as a starting material which contains the complete genetic information of an organism, and by extracting it, scientists can access and analyze this information. This information is essential for developing effective conservation strategies and understanding the complex processes of evolution. The objective of this study was to develop a protocol for extracting High-Quality genomic DNA from the arid tree species Prosopis cineraria. The current extraction protocol is based upon the conventional cetyl trimethyl ammonium bromide (CTAB) method with further modifications for the extraction of DNA. The principle modifications currently employed for DNA extraction involved the use of higher concentration of CTAB and sodium chloride. Additionally, L-ascorbic acid and Polyvinylpyrrolidone (PVP) was also added. The incubation time was doubled and DNA was precepted in 100% ice-cold ethanol. Further a RNAse treatment was also included for 30 mins. The yield of DNA ranged from 630-4200ng/µl with average yield of 2041ng/µl, and the absorbance lied between 1.6-2.15 for A260/280 and 1.64-2.31 for A260/230, indicating minimal levels of contaminating metabolites. This method offers a simple, efficient, and cost-effective way to extract high-quality genomic DNA, making it ideal for large-scale genetic studies.

Optimizing Genomic DNA Extraction from Avian Feathers: A Modified Phenol-Chloroform Approach for Enhanced Efficiency and Cost-Effectiveness.

The procurement of blood and tissue samples for DNA extraction in avian species intended for molecular studies is associated with the induction of discomfort and pain in the subjects, compounded by practical challenges in application and ethical considerations. Consequently, feathers have emerged as a more prevalent source for molecular investigations, particularly in the fields of poultry and ornithology. However, the effective extraction of DNA from feathers necessitates the breakdown of the hard keratinized tissue within the feather structure. This study aimed to devise a highly efficient, cost-effective, and easily adaptable Modified Phenol-Chloroform (MPC) approach for genomic DNA extraction from feathers, addressing shortcomings identified in previous studies on feather-based DNA isolation. The MPC method was employed to extract genomic DNA from feather samples obtained from six distinct avian species (chicken, guinea fowl, canary, pigeon, emu, and goose). Comparative evaluation of DNA isolation efficiency was conducted by employing two different commercial DNA kits alongside the MPC method. The results showed significantly higher DNA concentrations (ng/ml) from chicken feathers using the MPC method compared to those obtained with commercial kits (p < 0.05), along with high DNA purity (1.83 ± 0.11). Subsequent PCR experiments, employing nuclear and mitochondrial DNA-specific primers, illustrated the effective amplification of short and long fragments from MPC-isolated DNA samples. In contrast to commercial kits, the findings underscore the successful application of the MPC method in isolating high-quality genomic DNA from feathers characterized by elevated keratin content.

Forensic STR and SNP Genotyping of Formalin-Fixed Skeleton Samples With Illumina's ForenSeq System.

Formalin fixatives are widely used in forensics to preserve tissues. However, extracting high-quality genomic DNA from formalin-fixed samples is challenging. Traditional short tandem repeat (STR) analysis using capillary electrophoresis (CE) for forensic DNA typing frequently results in failure. Massively parallel sequencing (MPS) can handle many samples and thousands of genetic markers, usually single-nucleotide polymorphisms (SNPs) and STRs, in a single test. Thus, it is useful for assessing highly deteriorated forensic evidence. Few studies have examined the effectiveness of STRs and SNPs genotyping of formalin-fixed skeletons using MPS. In this study, 55 skeletal samples from 5 individuals that were treated under different formalin fixation times (5-75 days) were examined and sequenced using the ForenSeq DNA Signature Prep Kit on the Illumina MiSeq FGX platform. The results showed that as the duration of formalin fixation increased, the detection rates of STRs and SNPs gradually decreased. After 75 days of fixation, the average detection rates for STRs and SNPs were 4% and 10%, respectively. The cumulative discrimination power (CDP) of individual identification SNPs (iiSNPs) was >0.9999 on the 45th day. However, the CDP of STRs was 0.9930 on the 22nd day. Low detection rates were observed for six STRs (D1S1656, PentaE, D22S1045, PentaD, DX8378 and DX10103) and five SNPs (rs2920816, rs354439, rs1736442, rs338882 and rs1031825). In conclusion, DNA extracted from formalin-fixed skeletons decomposes rapidly over time, and MPS technology can be a useful tool for detecting forensic genetic markers in such samples.

B-185 A Comparison Between Two Oral Sample Collection Devices on Preserving High-Quality Genomic DNA

Abstract Background High-quality genomic testing significantly enhances the knowledge on gene-disease association, and greatly benefits future biomedical research and precision medicine. However, widely used saliva sample collection presents challenges with high-quality and quantity of human DNA and high bacterial DNA ratio. In this study, we validated the performance of two commercially available oral collection devices for their ability to preserve high-quality genomic DNA long-term at room temperature. Methods Fifty-one buccal samples in total from twelve donors were collected using two kinds of commercially available oral sample collection devices (iSWAB®-DNA (Device 1) and OCD-100 (Device 2)). All of the collected samples were stored at ambient temperature for up to 1 year. Collected samples were extracted with QIAamp Blood Mini Kit (Qiagen) at baseline, 30 days, 60 days, and 1 year. All DNA yields were measured using a Nanodrop One Microvolume UV-Vis Spectrophotometer; TapeStation System was used for determining the integrity of DNA. The bacterial DNA ratio was estimated by quantitative PCR with primers targeting the 16S rRNA gene known to be present in prokaryotes but not eukaryotes. Results Device 1 had higher yield (Device 1: 18.59 ± 8.98; Device 2: 6.44 ± 4.62), lower bacterial DNA ratio (Device 1: 2.06 ± 1.93; Device 2: 3.79 ± 4.58), and higher overall DNA integrity (Device 1: 6.1 ± 0.31; Device 2: 5.42 ± 0.96) compared to Device 2. Device 1 demonstrated longer post-collection sample stability at ambient temperature compared with Device 2 regarding DNA yield, bacterial DNA ratio and DNA integrity. No significant changes in DNA yield, integrity, and bacterial DNA ratio were observed after one year of storage in Device 1. No significant changes in DNA yield, integrity, and bacterial DNA ratio were observed after 60 days of storage in Device 2. However, Device 2 showed a significant increase in DNA yield, bacterial DNA ratio, and DNA integrity comparing baseline to 1 year. Conclusions Device 1 preserves higher quantity and quality of DNA from oral samples at ambient temperature for up to 1 year. These advantages significantly enhance efficiency and cost-effectiveness in downstream applications such as genotyping and sequencing, making it a valuable tool in genomics research with a commendable success rate.

B-184 Purifying High-Quality Genomic DNA From Frozen Blood Samples Stored up to 1.5 Years at -20°C

Abstract Background Maintaining DNA integrity in blood samples, from transport to storage to processing, is crucial to the success of downstream applications, especially regarding diagnostic applications. Additionally, due to the invasive nature of collecting blood samples, minimizing resampling to avoid excess patient pain and cost of collection and transportation are important to consider. Having the option to store and maintain portions of blood samples in the freezer enables future retesting without recollection. Since -20°C freezers generally are more affordable, require less energy, and have smaller footprints than -80°C freezers, demonstrating frozen blood stability at -20°C temperatures could decrease sampling burdens for labs with facility constraints. Methods Whole blood was collected into 9mL vacuum tubes (HemaSure-OMXP) from healthy donors. Aliquots of whole blood and buffy coat samples were stored at -20°C and thawed for genomic DNA extractions (QIAamp DNA Blood Mini Kit, Qiagen) at a range of different timepoints; frozen whole blood samples were processed at 0, 49, 141, and 148 days and frozen buffy coat samples were processed at 7, 27, 36, 185, 246, 373, 394, 584, and 604 days. Purified gDNA was quantified with Qubit (Broad Range dsDNA Assay, ThermoFisher) and qualified with Nanodrop One Microvolume UV-Vis Spectrophotometer (ThermoFisher) and TapeStation (Genomic DNA ScreenTape, Agilent Technologies). Results No significant decrease was observed for DNA yield and quality between fresh and frozen whole blood samples. Average DNA yields from 200 μL whole blood were 4.5 ± 2.2 μg and 5.3 ± 1.2 μg for fresh and frozen blood respectively. Average DNA integrity (DIN) was 6.9 ± 0.4 and 7.1 ± 0.3 for fresh and frozen blood respectively. Donor differences in blood samples can help explain the variance in DNA yields reported. No significant decrease was observed for DNA yield and quality in buffy coat samples frozen for longer at -20°C. In total, 84 whole blood and 25 buffy coat samples were processed in this study. Conclusions Although buffy coat samples tended to have higher yields than whole blood, due to the higher amount of white blood cells present, both sample types provided sufficient amounts of gDNA for downstream applications. Freezing blood samples for future testing can be effective at reducing the need for resampling without sacrificing the nucleic acid yield or quality of fresh samples. Additionally, as technological advancements are made for equipment and assays, frozen blood samples can be retested and compared to previous results for improved diagnostic decisions, done without requiring recollection from labs and patients.

High-Quality Genomic DNA Extraction Protocol for Bacillus and Clostridium Species.

High-quality DNA with sufficient yield is the goal of DNA extraction protocols. We present an optimized, cost-effective method for extracting next-generation sequencing (NGS)-quality genomic DNA from Bacillus and Clostridium species using the chloroform-isoamyl approach. The protocol involves two main procedures: cultivation of the bacteria under appropriate conditions, followed by DNA extraction through cell lysis, phase separation, DNA precipitation, and cleanup. This method has been successfully applied to several hundred strains of Bacillus and Clostridium species in our laboratory, including B. cereus, B. licheniformis, C. sporogenes, and C. tyrobutyricum, demonstrating its efficacy and reliability for producing high-quality DNA that meets NGS quality control standards. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing Bacillus and Clostridium species Basic Protocol 2: DNA extraction © 2024 by John Wiley & Sons, Inc.

GoEnrich: creating high quality genomic DNA resources from limited voucher specimen tissues or museum specimens of at-risk species for conservation-friendly use in the validation of environmental DNA assays

ObjectiveEnvironmental DNA (eDNA) methods are crucial for monitoring populations, particularly rare and cryptic species. For confident eDNA application, rigorous assay validation is required including specificity testing with genomic DNA (gDNA). However, this critical step is often difficult to achieve as obtaining fresh tissue samples from at-risk species can be difficult, highly limited, or impossible. Natural history museum collections could serve as a valuable and ethical voucher specimen resource for eDNA assay validation. The present study demonstrates the effectiveness of whole genome amplification (WGA) in providing enough gDNA to assemble high quality mitogenomes from which robust targeted eDNA assays can be designed.ResultsUsing fresh and historical museum tissue samples from six species spanning fish, birds, and mammals, we successfully developed a WGA method with an average yield of 380 to 1,268 ng gDNA per 20 µL reaction. This gDNA was used for whole genome shotgun sequencing and subsequent assembly of high quality mitogenomes using mtGrasp. These mitogenomes were then used to develop six new robust, targeted quantitative real time polymerase chain reaction-based eDNA assays and 200 ng WGA-enriched yielded satisfactory Cq values and near 100% detection frequencies for all assays tested. This approach offers a cost-effective and non-invasive alternative, streamlining eDNA research processes and aiding in conservation efforts.

A straightforward technique to obtain genomic DNA from nasal swabs suitable for sheep SNP genotyping analysis

Isolation of high quality and quantity genomic DNA is essential for molecular studies. It is crucial to select a non-invasive and straightforward technique to ensure the efficient collection of DNA, particularly at the farm level. The aim of this study was to determine if nasal swabs are an appropriate biological matrix to obtain good quality genomic DNA suitable for SNP genotyping. In this study, two biological matrices (blood and nasal swabs) were evaluated and compared for the isolation of genomic DNA obtained from 15 female Texel sheep. DNA quality and quantity were assessed using spectrophotometry and gel electrophoreses. Genotype concordance rates were used for comparison. Results showed that the highest concentration mean was obtained from blood samples (159.14 ng/µl), while from nasal swab samples the concentration mean was lower (130.12 ng/µl), but the difference was non-significant. Regarding purity, DNA obtained from nasal swabs presented a higher A260/A280 ratio (1.96), while the one obtained from blood samples was 1.90. Total DNA yield obtained from blood samples (15.91 µg) was significantly higher than the one obtained from nasal swabs (6.51). Blood and nasal swab genotyping concordance rates were high (mean = 0.984). In conclusion, our results indicate that nasal swabs can yield good quality DNA; however, the DNA extraction protocol should be optimized.

Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy

Abstract Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions. Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay. Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models. Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels. Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7248.

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 modified CTAB method for extracting high-quality genomic DNA from aquatic plants

This study introduces a streamlined approach for extractinghigh-quality DNA fromaquatic plants using CTAB, catering to molecular studies. Seven aquatic plant species(Hygrophilaauriculata, Limnophilarepens, Crinum malabaricum, Lagenandraovata, Ludwigiaperuviana, Eichhorniacrassipes, and Ipomoea aquatica) spanning six orderswere subjected toDNA extraction. The method combinesmechanical lysis and chemical treatments to effectively disrupt cells, coupled with RNase treatment and phenol extraction to mitigate RNA and protein contamination. The optimizedCTAB protocol facilitatesthe extraction of high-quality genomic DNA, suitable for amplifying plant barcode genes such as ITS and rbcL, as well asmarkerslike RAPD and ISSR, thereby enhancing the efficiency and reliability of genomic studies in aquatic plants.

An innovative optimized protocol for high-quality genomic DNA extraction from recalcitrant Shea tree (Vitellaria paradoxa, C.F. Gaertn) plant and its suitability for downstream applications.

It is not always easy to find a universal protocol for the extraction of genomic DNA (gDNA) from plants. Extraction of gDNA from plants such as shea with a lot of polysaccharides in their leaves is done in two steps: a first step to remove the polysaccharides and a second step for the extraction of the gDNA. In this work, we designed a protocol for extracting high-quality gDNA from shea tree and demonstrate its suitability for downstream molecular applications. Fifty milligrams of leaf and root tissues were used to test the efficiency of our protocol. The quantity of gDNA was measured with the NanoDrop spectrometer and the quality was checked on agarose gel. Its suitability for use in downstream applications was tested with restriction enzymes, SSRs and RAPD polymerase chain reactions and Sanger sequencing. The average yield of gDNA was 5.17; 3.96; 2.71 and 2.41µg for dry leaves, dry roots, fresh leaves and fresh roots respectively per 100mg of tissue. Variance analysis of the yield showed significant difference between all tissue types. Leaf gDNA quality was better compared to root gDNA at the absorbance ratio A260/280 and A260/230. The minimum amplifiable concentration of leaf gDNA was 1pg/µl while root gDNA remained amplifiable at 10pg/µl. Genomic DNA obtained was also suitable for sequencing. This protocol provides an efficient, convenient and cost effective DNA extraction method suitable for use in various vitellaria paradoxa genomic studies.

Genomic DNA polymorphism in &lt;i&gt;Myrica gale&lt;/i&gt; (Myricaceae) in the Lebyazhiy state Nature Reserve (the southern coast of the gulf of Finland)

The analysis of genomic DNA polymorphism is one of widely used approaches for studying the genetic structure of natural populations. It has been successfully applied to various plants. However, many species have not yet been studied, which is primarily due to methodological difficulties in isolating well-purified and non-degraded genomic DNA. These difficulties are due to the fact that plants possess numerous bioorganic compounds (polysaccharides, polyphenols, lipids, etc.) that contaminate DNA and significantly reduce its quality. Such species include marsh waxweed (Myrica gale L.), a perennial sub-Atlantic shrub with presumably vegetative propagation (in nature, waxweed seedlings are quite rare). We developed a simple protocol for isolation of high-quality genomic DNA from waxweed leaves and performed AFLP analysis of 42 plants of this species from three subpopulations in the Lebyazhiy Nature Reserve. Using three primer pairs, we isolated 22 amplification fragments, 8 of which were monomorphic. For the remaining 14 fragments, the average level of their polymorphism was low: depending on the subpopulation studied, it varied from 0.079 to 0.129. As shown by our analysis, all three studied subpopulations are polymorphic with a predominance of two common AFLP-genotypes. The corresponding plants are apparently the vegetative descendants of the founders. The rare AFLP-genotypes (represented by just one or two plants; a total of 12 such genotypes were identified) are likely the result of mutational and recombination processes. Our data give evidence that in the life cycle and dispersal of waxweed, the role of sexual reproduction is also noticeable.

SarCTAB: an efficient and cost-effective DNA isolation protocol from geophytes.

Geophytes are herbaceous plants that grow anew from underground buds and are excellent models to study storage organ formation. However, molecular studies involving geophytes are constrained due to the presence of a wide spectrum of polysaccharides and polyphenols that contaminate the genomic DNA. At present, several protocols exist for the extraction of genomic DNA from different plant species; however, isolating high-quality DNA from geophytes is challenging. Such challenges are further complexed by longer incubation time and multiple precipitation steps involved in existing DNA isolation methods. To overcome such problems, we aimed to establish a DNA extraction method (SarCTAB) which is an economical, quick, and sustainable way of DNA isolation from geophytes. We improved the traditional CTAB method by optimizing key ingredients such as sarcosine, β-mercaptoethanol, and high molar concentration of sodium chloride (NaCl), which resulted in high concentration and good-quality DNA with lesser polysaccharides, proteins, and polyphenols. This method was evaluated to extract DNA from storage organs of six different geophytes. The SarCTAB method provides an average yield of 1755ng/µl of high-quality DNA from 100mg of underground storage tissues with an average standard purity of 1.86 (260/280) and 1.42 (260/230). The isolated genomic DNA performed well with Inter-simple sequence repeat (ISSR) amplification, restriction digestion with EcoRI, and PCR amplification of plant barcode genes viz. matK and rbcL. Also, the cost involved in DNA isolation was low when compared to that with commercially available kits. Overall, SarCTAB method works effectively to isolate high-quality genomic DNA in a cost-effective manner from the underground storage tissues of geophytes, and can be applied for next-generation sequencing, DNA barcoding, and whole genome bisulfite sequencing.

Genomic DNA extraction from the medicinal plant Crocus sativus : Optimization of Standard Methods

High-quality genomic DNA is essential for genomic and molecular investigations such as next-generation sequencing. However, DNA extraction from medicinal plants like Crocus sativus can be challenging due to their high secondary metabolite content, which can interact with nucleic acids and affect the quality and yield of extraction. This study aimed to optimize the quality and yield of DNA using the cetyltrimethylammonium bromide (CTAB) extraction method from the leaves, stigma, and saffron corm. This new method is easy to use and can be performed using standard equipment and inexpensive reagents. The modifications made to the CTAB lysis buffer in this study, with the addition of SDS, resulted in a yield of 4233 ng/µl of DNA per sample of saffron corm (100 mg). This protocol is efficient and cost-effective for DNA extraction for studies with large samples and limited resources. This method is expected to be widely used for large-scale plant extraction and has a broad application in PCR-based sequencing studies.

A modified DNA isolation protocol for high-quality DNA and long-term storability in grasspea (Lathyrus sativus L.)

Grasspeas pose challenges in obtaining high-quality DNA due to their seed and leaves’ high protein, phenols, secondary metabolites, and neurotoxins. This study investigated the challenges faced by the CTAB method in DNA isolation, subsequent enhancements in quality and storage with a modified CTAB approach using triton, PVP, high salt TE and sodium acetate to extract the high-quality genomic DNA from grasspea.

An optimal genomic DNA extraction method for shoots of four Dendrocalamus species based on membership function analysis.

High-quality genomic DNA extraction is fundamental for the study of gene cloning and expression in plants. Therefore, this study evaluated several methods for extracting genomic DNA from shoots of four Dendrocalamus species to determine the optimal technique. Genomic DNA was extracted using three different methods: a commercial DNA extraction kit method, a modified cetyltrimethylammonium bromide method and a sodium dodecyl sulfate method. A membership function analysis was employed to compare these methods. The results demonstrated that the commercial DNA extraction kit method was the most effective and comprehensive approach for extracting genomic DNA from shoots of four Dendrocalamus species. Furthermore, this study provided valuable insights into optimizing techniques for extracting genomic DNA in other bamboo species.

Cationic and anionic detergent buffers in sequence yield high-quality genomic DNA from diverse plant species

Because of the heterogeneity among seedlings of outbreeding species, the use of seedling tissues as a source of DNA is unsuitable for the genomic characterization of elite germplasms. High-quality DNA, free of RNA, proteins, polysaccharides, secondary metabolites, and shearing, is mandatory for downstream molecular biology applications, especially for next-generation genome sequencing and pangenome analysis aiming to capture the complete genetic diversity within a species. The study aimed to accomplish an efficient protocol for the extraction of high-quality DNA suitable for diverse plant species/tissues. We describe a reliable, and consistent protocol suitable for the extraction of DNA from 42 difficult-to-extract plant species belonging to 33 angiosperm (monocot and dicot) families, including tissues such as seeds, roots, endosperm, and flower/fruit tissues. The protocol was first optimized for the outbreeding recalcitrant trees viz., Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera, which are rich in proteins, polysaccharides, and secondary metabolites, and the quality of the extracted DNA was confirmed by downstream applications. Nine procedures were attempted to extract high-quality, impurities-free DNA from these three plant species. Extraction of the ethanol-precipitated DNA from cetyltrimethylammonium bromide (CTAB) protocol using sodium dodecyl sulfate (SDS) buffer, i.e., the extraction using a cationic (CTAB) detergent followed by an anionic (SDS) detergent was the key for high yield and high purity (1.75–1.85 against A260/280 and an A260/230 ratio of >2) DNA. A vice versa extraction procedure, i.e., SDS buffer followed by CTAB buffer, and also CTAB buffer followed by CTAB, did not yield good-quality DNA. PCR (using different primers) and restriction endonuclease digestion of the DNA extracted from these three plants validated the protocol. The accomplishment of the genome of P. cineraria using the DNA extracted using the modified protocol confirmed its applicability to genomic studies. The optimized protocol successful in extracting high-quality DNA from diverse plant species/tissues extends its applicability and is useful for accomplishing genome sequences of elite germplasm of recalcitrant plant species with quality reads.

Comprehensive analysis of the mitochondrial genome of Ziziphus mauritiana Lam.: Investigating sequence structure, repeat-mediated recombination, chloroplast-derived transfer, RNA editing, and evolution

Ziziphus mauritiana Lam. is a valuable semi-deciduous tropical fruit tree with economic, medicinal, nutritional, and environmental significance. While the chloroplast genome of Z. mauritiana has been studied, the decoding of its mitochondrial genome (mtGenome) is crucial for various research areas, including molecular phylogeny and functional genomics. In this study, we extracted high-quality genomic DNA from the green leaves of Z. mauritiana and assembled the mtGenome by integrating sequencing data from both long and short reads. The Z. mauritiana mtGenome spans 324,124 bp with a 45.43 % GC content and exhibits six distinct conformations alongside the main circular sequence due to three pairs of repetitive fragments that facilitate recombination. RNA editing events resulted in changes in the physicochemical properties of amino acids, with genes expressed at higher levels showing an increased frequency of editing sites. Chloroplast-to-mitochondria horizontal gene transfers occurred in Z. mauritiana; however, these transferred fragments had few mutations, posing a challenge in determining the exact timing of these transfers. Phylogenetic analysis highlights a slower evolution rate of mitochondrial genes compared to chloroplast genes. These insights greatly enhance our understanding of the dynamics of the Z. mauritiana mtGenome, including recombination, gene expression, evolutionary processes, and more. The data and results of this study will serve as valuable mtGenome data resources within the Rhamnaceae family, thereby facilitating further genomics-based studies on Z. mauritiana.

Physical Pretreatments Applied in Three Commercial Kits for the Extraction of High-Quality DNA from Activated Sewage Sludge.

Obtaining sufficient and high-quality genomic DNA from sludge samples is a fundamental issue of feasibility and comparability in genomic studies of microbial diversity. Commercial kits for soil are often used for the extraction of gDNA from sludge samples due to the lack of specific kits. However, the evaluation of the performance of commercial kits for sludge DNA extraction is scarce and optimization of these methods to obtain a high quantity and quality of DNA is necessary, especially for downstream genomic sequencing. Sequential batch reactors (SBRs) loaded with lignocellulosic biomass are used for the synthesis of renewable resources such as levulinic acid (LA), adipic acid (AA), and polyhydroxyalkanoates (PHAs), and the biochemical synthesis of these compounds is conducted through the inoculation of microbes present in the residual activated sludge (AS) obtained from a municipal wastewater treatment plant. To characterize these microbes, the extraction of DNA from residual sewage sludge was conducted with three different commercial kits: Nucleospin® Soil from Macherey-Nagel, DNEasy® PowerSoil® from Qiagen, and E.Z.N.A.® Plant DNA Kit from Omega BIO-TEK. Nevertheless, to obtain the highest load and quality of DNA for next-generation sequencing (NGS) analysis, different pretreatments and different combinations of these pretreatments were used. The pretreatments considered were an ultrasonic bath and a temperature of 80 °C, together and separately with different incubation time periods of 30, 60, and 90 min. The results obtained suggest a significant improvement in the efficiency and quality of DNA extraction with the three commercial extraction kits when used together with the ultrasonic bath and 80 °C for 60 min. Here, we were able to prove that physical pretreatments are a viable alternative to chemical lysis for DNA extraction from complex samples such as sludge.