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Genomic resources have yielded unprecedented insights into ecological and evolutionary processes, not to mention their importance in economic and conservation management of specific organisms. However, the field of macroalgal genomics is hampered by difficulties in the isolation of suitable DNA. Even when DNA that appears high quality by standard metrics has been isolated, such samples may not perform well during the sequencing process. We here have compared Oxford Nanopore long‐read sequencing results for three species of macroalgae to those of nonmacroalgal species and determined that when using macroalgal samples, sequencing activity declined rapidly, resulting in reduced sequencing yield. Chemical analysis of macroalgal DNA that would be considered suitable for sequencing revealed that DNA derived from dried macroalgae was enriched for polyphenol–DNA adducts (DNA with large polyphenols chemically attached to it), which may have led to sequencing inhibition. Of note, we observed the strongest evidence of sequencing inhibition and reduced sequence output when using samples dried using silica gel—suggesting that such storage approaches may not be appropriate for samples destined for Oxford Nanopore sequencing. Our findings have wide‐ranging implications for the generation of genomic resources from macroalgae and suggest a need to develop new storage methods that are more amenable to Oxford Nanopore sequencing or to use fresh flash‐frozen tissue wherever possible for genome sequencing. [ABSTRACT FROM AUTHOR]

BACKGROUND RESULTS CONCLUSION The identification of adulterated sweet potato vermicelli faces significant challenges, seriously hindering the development of the vermicelli industry. Herein, we investigate effects of energy‐gathered ultrasound (EGU) and energy‐divergent ultrasound (EDU) (30, 40 and 50 W  L−1) on structure, DNA extraction and adulterated quantification of sweet potato vermicelli and its starch, thereby exploring their potential in adulteration of sweet potato vermicelli.EGU‐assisted modified cetyltrimethylammonium bromide (CTAB) protocol with β‐mercaptoethanol significantly improved DNA extraction from sweet potato vermicelli (223.7–249.2 ng μL−1) and its starch (133.4–186.4 ng μL−1), followed by EDU‐assisted DNA extraction from sweet potato vermicelli (115.1–209.3 ng μL−1) and its starch (33.4–61.0 ng μL−1). Both EGU and EDU treatments resulted in the destruction of microstructure and crystalline structure, as well as changes in pasting and thermal properties of sweet potato vermicelli and its starch. Real‐time polymerase chain reaction (PCR) amplification results revealed that EGU and EDU enhanced the efficiency of DNA amplification, and EDU showed smaller cycle threshold (Ct) values than EGU. In addition, EDU‐assisted CTAB protocol combined with real‐time PCR could detect levels of less than 1% of cassava and maize starches in sweet potato vermicelli.EDU‐assisted CTAB protocol combined with real‐time PCR shows promise as a sensitive and reliable analytical tool for vermicelli adulteration. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Recent reports have disclosed the occurrence of Wolbachia in Aedes aegypti. Our study detected Wolbachia infection in Ae. aegypti by screening wild adult mosquitoes using two Wolbachia‐specific molecular markers. Overall, 444 adult Ae. aegypti mosquitoes were collected from April 2022 to October 2022 in Jeddah, Saudi Arabia. Each individual sample was processed and screened for the presence of Wolbachia using selected markers, the Wolbachia‐specific 16S rDNA and the Wolbachia surface protein gene (wsp), under optimized polymerase chain reaction (PCR) conditions, and sequenced. In total, 39 (8.78%) and 48 (10.81%) individual mosquito samples were determined to be infected with Wolbachia using the wsp and 16S rDNA markers, respectively. By utilizing two Wolbachia‐specific molecular markers, our study demonstrated the presence of Wolbachia from individual Ae. aegypti samples. Our results showed a low rate of Wolbachia infection and inferred that the detected strain belongs to supergroup B. [ABSTRACT FROM AUTHOR]

Efforts to characterize microbial life across diverse environments have progressed tremendously, yet the microbiome of Earth's largest biomass reservoir—the wood of living trees—has been largely unexplored. Current understanding of the tree microbiome is largely confined to roots and leaves, with little attention given to the endophytic microbiome of wood, even though emergent studies have indicated this zone as a niche for unique taxa, of consequence for ecosystem health and global biogeochemical cycles. The lack of investigation derives partly from the physical recalcitrance of wood, which presents challenges during sampling, homogenization, and the extraction of nucleic acids.In response to these issues, we present an optimized method for processing wood for use in microbial analyses, from sampling through to downstream analyses.Using methane‐cycling taxa as model endophytes, we assess losses in recovery during our method, and determine a limit‐of‐detection of approximately 500 cells per 100 mg of (dry) wood. For all six species evaluated—which represented several diverse taxa of hardwoods and softwoods—PCR inhibition proved minimal, and we expect this method to be applicable for a majority of tree species.The methods presented herein can facilitate future investigation into the wood microbiome and global microbial ecology of methane cycling. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) is used for biodiversity assessments in a variety of ecosystems across the globe, whereby different eDNA concentration, preservation and extraction methods can outperform others depending on the sampling conditions and environment. Tropical and subtropical ecosystems in Africa are among the less studied systems concerning eDNA‐based monitoring. Waterholes in arid parts of southern Africa represent important agglomeration points for terrestrial mammals, and the eDNA shed into such waterbodies provides a powerful source of information for monitoring mammalian biodiversity in the surrounding area. However, the applied methods for eDNA sampling, preservation and filtering in different freshwater systems vary greatly, and rigorous protocol testing in African freshwater systems is still lacking. This study represents the first attempt to examine variations in eDNA concentration, preservation and extraction methods under remote field conditions using waterborne eDNA in a savanna system. Collected samples were heavily affected by microalgal and bacterial growth, impeding eDNA capture and PCR success. We demonstrate clear effects of the methodological choices, which also depend on the state of eDNA. A preliminary metabarcoding run showed little taxonomic overlap in mammal species detection between two metabarcoding primers tested. We recommend water filtering (using filters with pore sizes >1 μm) over centrifugation for eDNA concentration, Longmire's solution for ambient temperature sample preservation and Qiagen's DNeasy PowerSoil Pro Kit for DNA extraction of these inhibitor‐prone samples. Furthermore, at least two independent metabarcoding markers should be utilized in order to maximize species detections in metabarcoding studies. [ABSTRACT FROM AUTHOR]

Background: The analysis of genetically modified organisms (GMOs) in food and feed is broadly based on the detection of target DNA sequences using PCR‐based methods. The first step of a typical workflow in a GMO testing laboratory is DNA extraction and purification. Several DNA extraction methods have been described for this purpose so far. Whatever the method adopted, the yield and the quality of the extracted DNA are essential factors for the success of GMO analysis. Results: The DNA extraction yield was variable between the different methods, while quality and efficiency appeared comparable. Each method allowed a different yield and a slight correlation between inhibition and the food/feed matrix extracted was observed. Particularly for challenging matrices, the method used for DNA extraction has a remarkable influence on both the quality and quantity of the recovered DNA. Small variations in value % GM content were observed in complex, heterogeneous matrices but not in CRMs and homogeneous matrices. Conclusions: In this study, the purpose is not to establish the best method of extraction but to examine several parameters that play an important role in GMO detection and verify a possible effect on the quantified GMO percentage. In particular, it was found that the yield and quality of extracted DNA depends on the type of sample, notably the degree of processing, composition, particle size, and the protocol used, especially in relation to sample intake. The slightly different GM content observed in heterogeneous matrices could raise doubt in the case of samples close to the legal threshold. [ABSTRACT FROM AUTHOR]

Warts, caused by human papillomavirus (HPV) infection, have various clinical presentations, making them difficult to differentiate from clavus, callus, and sometimes, squamous cell carcinoma. Although skin biopsies are the gold standard, a less‐invasive method of examining these lesions is desired. Ninety patients with warts and related diseases, such as clavus and callus, were recruited to explore new differentiation methods using the surface of the warts. DNA was extracted from three types of specimens in each case: surface swab, shaved hyperkeratotic scale, and post‐shaved surface swab. Total DNA was successfully extracted from these three specimens and was sufficient for subsequent HPV DNA detection. We analyzed samples for the HPV type and HPV viral load using polymerase chain reaction (PCR). Fifty‐five cases were PCR‐positive, and HPV1a, 2a, 4, 27, 57, and 65 were detected. The amount of HPV1a DNA produced was significantly greater than that of other HPV types. Regarding the correlation between the clinical diagnosis and HPV detection, the positive agreement rate was 90.9%, the negative agreement rate was 40.0%, and the overall agreement rate was 71.1%. Ten of the 21 cases clinically diagnosed as plantar warts were PCR‐negative, especially in elderly patients. This suggests that it is difficult to distinguish plantar warts from clavus and callus in clinical practice. Although the amount of HPV DNA in the removed keratinization scale was highest for all HPV types, HPV detection by swabbing before and after shaving is also useful for follow‐up as well as for differential diagnosis. [ABSTRACT FROM AUTHOR]

Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high‐quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high‐throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane‐based spin columns and a 96‐well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96‐well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead‐beating procedure, which optimizes altogether the cost, handling time and bacteria‐specific effects associated with enzymatic lysis. [ABSTRACT FROM AUTHOR]

Next‐generation sequencing technology has enabled accurate insights into the diet of wildlife species. The protocols for faecal sample collection and DNA extraction for diet analysis have differed from those focusing on target species, even in most studies combining questions on both aspects. We designed an experiment to evaluate two protocols using 11 parameters and select a single one that will generate both target species (Asiatic wild ass, Equus hemionus, in Israel) and diet DNA, as an effective strategy to minimise time, effort, and cost without hampering efficiency. In Protocol A, we swabbed the outer surface of faecal boluses and extracted DNA using a Stool Kit, while for Protocol B, we homogenised faecal matter from inside the bolus followed by extraction using a Powersoil Kit. Protocol A performed significantly better for four parameters, which included, for the target species, microsatellite amplification success and the quantity of the GAPDH gene; and for its diet, the number of exact sequence variants (ESVs) obtained at genus level and plant genus richness. However, there was no significant difference in the amplification success of sex‐linked and plant markers, total reads at genus level, number of genera obtained and plant genus composition. Although we chose Protocol A, both protocols yielded results for the target species and its diet, demonstrating that one single protocol can be used for both purposes, although a pilot study is recommended to optimise the protocol for specific systems. This strategy may also be useful for studies combining target species and their gut microbiome and parasitic load. [ABSTRACT FROM AUTHOR]

Premise: Plants with stiff, leathery leaves pose a challenge for standard DNA extraction protocols. These tissues are recalcitrant to mechanical disruption via TissueLyser (or analogous devices) and are often high in secondary metabolites. These compounding factors result in low yields, which may be sufficient for PCR amplification but are generally inadequate for genomic applications that require large quantities of high‐quality DNA. Cycads in the genus Encephalartos exemplify these challenges, as this group of plants is fortified for life in harsh, dry habitats with notoriously thick and rigid leaves. Methods and Results: Using a DNA extraction kit, we tested three methods of mechanical disruption and examined the differences between stored vs. freshly collected samples and mature vs. senescing leaflets. We found that the manual method of pulverizing tissue yields the highest concentrations of DNA, and that both senescing leaflets and leaflet tissue that has been stored for extended periods yield sufficient DNA for genomic analyses. Conclusions: These findings shed light on the feasibility of using senescing leaves and/or tissue that has been stored on silica for long periods of time when attempting to extract large amounts of DNA. We provide here an optimized DNA extraction protocol that can be applied to cycads and other plant groups with tough or rigid leaves. [ABSTRACT FROM AUTHOR]

Premise: A novel protocol for rapid plant DNA extraction using microneedles is proposed, which supports botanic surveys, taxonomy, and systematics. This protocol can be conducted in the field with limited laboratory skills and equipment. The protocol is validated by sequencing and comparing the results with QIAGEN spin‐column DNA extractions using BLAST analyses. Methods and Results: Two sets of DNA extractions were conducted on 13 species spanning various leaf anatomies and phylogenetic lineages: (i) fresh leaves were punched with custom polymeric microneedle patches to recover genomic DNA, or (ii) QIAGEN DNA extractions. Three plastid (matK, rbcL, and trnH‐psbA) and one nuclear ribosomal (ITS) DNA regions were amplified and sequenced using Sanger or nanopore technology. The proposed method reduced the extraction time to 1 min and yielded the same DNA sequences as the QIAGEN extractions. Conclusions: Our drastically faster and simpler method is compatible with nanopore sequencing and is suitable for multiple applications, including high‐throughput DNA‐based species identifications and monitoring. [ABSTRACT FROM AUTHOR]

Premise: Developing an effective and easy‐to‐use high‐molecular‐weight (HMW) DNA extraction method is essential for genomic research, especially in the era of third‐generation sequencing. To efficiently use technologies capable of generating long‐read sequences, it is important to maximize both the length and purity of the extracted DNA; however, this is frequently difficult to achieve with plant samples. Methods and Results: We present a HMW DNA extraction method that combines (1) a nuclei extraction method followed by (2) a traditional cetyltrimethylammonium bromide (CTAB) DNA extraction method for plants with optimized extraction conditions that influence HMW DNA recovery. Our protocol produced DNA fragments (percentage of fragments >20 kbp) that were, on average, ca. five times longer than those obtained using a commercial kit, and contaminants were removed more effectively. Conclusions: This effective HMW DNA extraction protocol can be used as a standard protocol for a diverse array of taxa, which will enhance plant genomic research. [ABSTRACT FROM AUTHOR]

Cetyltrimethylammonium bromide (CTAB)–based methods are widely used to isolate DNA from plant tissues, but the unique chemical composition of secondary metabolites among plant species has necessitated optimization. Research articles often cite a "modified" CTAB protocol without explicitly stating how the protocol had been altered, creating non‐reproducible studies. Furthermore, the various modifications that have been applied to the CTAB protocol have not been rigorously reviewed and doing so could reveal optimization strategies across study systems. We surveyed the literature for modified CTAB protocols used for the isolation of plant DNA. We found that every stage of the CTAB protocol has been modified, and we summarized those modifications to provide recommendations for extraction optimization. Future genomic studies will rely on optimized CTAB protocols. Our review of the modifications that have been used, as well as the protocols we provide here, could better standardize DNA extractions, allowing for repeatable and transparent studies. [ABSTRACT FROM AUTHOR]

Premise: The use of cetyltrimethylammonium bromide (CTAB) is an effective and inexpensive method of extracting DNA from plants. The CTAB protocol is frequently modified to optimize DNA extractions, but experimental approaches rarely perturb a single variable at a time to systematically infer their effect on DNA quantity and quality. Methods and Results: We investigated how chemical additives, incubation temperature, and lysis duration affected DNA quantity and quality. Altering those parameters influenced DNA concentrations and fragment lengths, but only extractant purity was significantly affected. CTAB and CTAB plus polyvinylpyrrolidone buffers produced the highest DNA quality and quantity. Extractions from silica gel–preserved tissues had significantly higher DNA yield, longer DNA fragments, and purer extractants compared to herbarium‐preserved tissues. Conclusions: We recommend DNA extractions of silica gel–preserved tissues that include a shorter and cooler lysis step, which results in purer extractions compared to a longer and hotter lysis step, while preventing fragmentation and reducing time. [ABSTRACT FROM AUTHOR]

Premise: The objective of this study was to optimize an existing DNA extraction protocol for recalcitrant plant taxa to obtain high‐quality DNA from preserved herbarium tissue suitable for downstream PCR applications. Methods and Results: Leaf tissue from 30 diverse plant species was obtained from the U.S. National Arboretum Herbarium. Our previous DNA extraction protocol (Gouker et al., 2020, Applications in Plant Sciences 8: e11403) was improved by use of 10X Tris‐EDTA buffer, addition of polyvinylpolypyrrolidone, and omission of sample heating during homogenization, and resulted in total DNA yields ranging from 60–2460 ng. Optimized PCR amplification using universal plant primers for the ITS‐p3/u4 region and the P6 loop of the trnL (UAA) chloroplast intron was successful for most specimens. Conclusions: This protocol, which is simple, fast, and uses standard laboratory‐grade chemicals, yields DNA from herbarium specimens that is comparable in quality to that from commercially available kits, and is of sufficient quality and quantity for other applications. [ABSTRACT FROM AUTHOR]

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high‐yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non‐probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un‐insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express‐96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high‐throughput, high‐yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high‐yield and high‐quality DNA from compromised bone, teeth, and hair shaft samples. [ABSTRACT FROM AUTHOR]

An archive of 5 years of cases involving the identification of human remains was curated, collecting information on: The sample type submitted, the number of STR loci yielding interpretable results, the kinship challenge posed, and the outcome for the case. A total of 129 cases of remains ID were investigated using manual DNA extraction and recovery methods with amplification of STR markers using the Power Plex 21 multiplex STR kit from Promega Corp. In 52 cases, blood spots collected by the ME were provided as sample and in 100% of those cases, probabilities of relatedness to the reference samples was ≥99%. In 77 cases, tissue other than blood was provided as a source of DNA. These other samples were grouped categorically into long bones (femur and tibia; 40 cases), skull bones/teeth (11 cases), other bones (16 cases), and tissue (normally adherent to bone) (10 cases). Reference samples provided for cases included alleged parents or child(ren) of the victim (86 cases), alleged full siblings of the victim (38 cases), or alleged second‐order relatives (five cases). The overall success rate in confirming the identity of the source of the remains in these cases was 89.2%. Our results demonstrate that a laboratory can be often successful identifying human remains using methods easily implemented in any DNA typing laboratory. [ABSTRACT FROM AUTHOR]

Studies of parasites in wild animal populations often rely on molecular methods to both detect and quantify infections. However, method accuracy is likely to be influenced by the sampling approach taken prior to nucleic acid extraction. Avian Haemosporidia are studied primarily through the screening of host blood, and a range of storage mediums are available for the short‐ to long‐term preservation of samples. Previous research has suggested that storage medium choice may impact the accuracy of PCR‐based parasite detection, however, this relationship has never been explicitly tested and may be exacerbated by the duration of sample storage. These considerations could also be especially critical for sensitive molecular methods used to quantify infection (qPCR). To test the effect of storage medium and duration on Plasmodium detection and quantification, we split blood samples collected from wild birds across three medium types (filter paper, Queen's lysis buffer, and 96% ethanol) and carried out DNA extractions at five time points (1, 6, 12, 24, and 36 months post‐sampling). First, we found variation in DNA yield obtained from blood samples dependent on their storage medium which had subsequent negative impacts on both detection and estimates of Plasmodium copy number. Second, we found that detection accuracy (incidence of true positives) was highest for filter‐paper‐stored samples (97%), while accuracy for ethanol and Queen's lysis buffer‐stored samples was influenced by either storage duration or extraction yield, respectively. Lastly, longer storage durations were associated with decreased copy number estimates across all storage mediums; equating to a 58% reduction between the first‐ and third‐year post‐sampling for lysis‐stored samples. These results raise questions regarding the utility of standardizing samples by dilution, while also illustrating the critical importance of considering storage approaches in studies of Haemosporidia comparing samples subjected to different storage regimes and/or stored for varying lengths of time. [ABSTRACT FROM AUTHOR]

Premise: Sample preparation in genomics is a critical step that is often overlooked in molecular workflows and impacts the success of downstream genetic applications. This study explores the use of a recently developed focused ultrasound extraction (FUSE) technique to enable the rapid release of DNA from plant tissues for genetic analysis. Methods: FUSE generates a dense acoustic cavitation bubble cloud that pulverizes targeted tissue into acellular debris. This technique was applied to leaf samples of American chestnut (Castanea dentata), tulip poplar (Liriodendron tulipifera), red maple (Acer rubrum), and chestnut oak (Quercus montana). Results: We observed that FUSE can extract high quantities of DNA in 9–15 min, compared to the 30 min required for control DNA extraction methods. FUSE extracted DNA quantities of 24.33 ± 6.51 ng/mg and 35.32 ± 9.21 ng/mg from American chestnut and red maple, respectively, while control methods yielded 6.22 ± 0.87 ng/mg and 11.51 ± 1.95 ng/mg, respectively. The quality of the DNA released by FUSE allowed for successful amplification and next‐generation sequencing. Discussion: These results indicate that FUSE can improve DNA extraction efficiency for leaf tissues. Continued development of this technology aims to adapt to field‐deployable systems to increase the cataloging of genetic biodiversity, particularly in low‐resource biodiversity hotspots. [ABSTRACT FROM AUTHOR]

DNA metabarcoding is routinely used for biodiversity assessment, in particular targeting highly diverse groups for which limited taxonomic expertise is available. Various protocols are currently in use, although standardization is key to its application in large‐scale monitoring. DNA metabarcoding of arthropod bulk samples can be conducted either destructively from sample tissue, or nondestructively from sample fixative or lysis buffer. Nondestructive methods are highly desirable for the preservation of sample integrity but have yet to be experimentally evaluated in detail. Here, we compare diversity estimates from 14 size‐sorted Malaise trap samples processed consecutively with three nondestructive approaches (one using fixative ethanol and two using lysis buffers) and one destructive approach (using homogenized tissue). Extraction from commercial lysis buffer yielded comparable species richness and high overlap in species composition to the ground tissue extracts. A significantly divergent community was detected from preservative ethanol‐based DNA extraction. No consistent trend in species richness was found with increasing incubation time in lysis buffer. These results indicate that nondestructive DNA extraction from incubation in lysis buffer could provide a comparable alternative to destructive approaches with the added advantage of preserving the specimens for postmetabarcoding taxonomic work but at a higher cost per sample. [ABSTRACT FROM AUTHOR]

As technologies advance alongside metabarcoding and metagenomic resources, particularly for larger fungal genomes, DNA extraction methods must be optimized to meet higher thresholds, especially from complex environmental substrates. This study focused on extracting fungal genomic compounds from woody substrates, a challenge due to the embedment of endophytic and saprotrophic fungi within wood cells, the physical recalcitrance of wood, the adsorption of nucleic acids to wood polymers, and the release of downstream inhibitors. Hypothesizing that cetyltrimethylammonium bromide would be the best option, we compared prominent methods by extracting and sequencing microbial DNA from sound and decayed birch (Betula papyrifera) and pine (Pinus resinosa). DNA quantities varied significantly depending on extraction methods and decay stage. The quality of DNA, in terms of purity and integrity, significantly impacted whether the samples could be amplified and sequenced. However, amplicon sequencing of bacterial and fungal communities revealed no significant extraction bias. This, along with the sequencing effectiveness and cost/time efficiency, indicates that Qiagen is the gold standard for woody substrates. This study increases confidence in published amplicon data sets regardless of the extraction methods, provides a cost-benefit table for making protocol decisions, and offers guidance on fungal DNA extractions from complex organic substrates (sound and decayed wood) that would best suit future metagenomic efforts., (© 2024 The Author(s). MicrobiologyOpen published by John Wiley & Sons Ltd.)

Premise: Sorghum is a multi‐use crop, the efficient breeding of which requires the development of new genetic tools. One such tool could be the genetic assessment of free microspores, which are released just after the tetrad stage of pollen development. Microspores are ideal for DNA isolation as they have underdeveloped cell walls and can be readily lysed as natural protoplasts. Methods: Four cultivars of Sorghum bicolor (ʻAchi Turi', ʻDale', ʻLocal', and ʻTopper 76‐6') were grown in a greenhouse until flowering (7.7–11.5 cm flag leaf internode length), after which 30 immature microspores were isolated from each line. Plant height, time to flowering, boot radius, and spikelet maturation were recorded for each cultivar. The exine development of the microspores was observed under an inverted Nikon microscope, and those with underdeveloped exine were subjected to whole‐genome amplification and sequencing. Results: Microspores in the early uninucleate to early binucleate stages had underdeveloped exine, and were therefore ideal for DNA extraction. High‐quality DNA was obtained from these single‐cell gametophytes. The average DNA concentration was 2902 ng/µL, with fragment sizes comparable to those obtained from leaf tissue extractions. Discussion: Harvesting panicles with immature microspores means the entire gametic population is accessible for DNA analyses. This is the first amplification of whole‐genome DNA fragments from sorghum single‐cell microspores isolated during gametogenesis. [ABSTRACT FROM AUTHOR]

With increased application of DNA metabarcoding in biodiversity assessment, various laboratory protocols have been optimized, and their further evaluation is subject of current research. Homogenization of bulk samples and subsequent DNA extraction from a subsample of destructed tissue is a common first stage of the metabarcoding process. This can either be conducted using sample material soaked in a storage fixative, e.g., ethanol (here referred to as "wet" treatment) or from dried individuals ("dry"). However, it remains uncertain if perfect mixing and equal distribution of DNA within the tube is ensured during homogenization and to what extent incomplete mixing and resulting variations in tissue composition affect diversity assessments if only a fraction of the destructed sample is processed in the downstream metabarcoding workflow. Here we investigated the efficiency of homogenization under wet and dry conditions and tested how variations in destructed tissue composition might affect diversity assessments of complex arthropod samples. We considered five time intervals of Malaise trap bulk samples and process nine different subsamples of homogenized tissue (20 mg each) in both treatments. Results indicate a more consistent diversity assessment from dried material, but at the cost of a higher processing time. Both approaches detected comparable OTU diversity and revealed similar taxa compositions in a single tissue extraction. With an increased number of tissue subsamples during DNA extraction, OTU diversity increased for both approaches, especially for highly diverse samples obtained during the summer. Here, particularly the detection of small and low‐biomass taxa increased. The processing of multiple subsamples in the metabarcoding protocol can therefore be a helpful procedure to enhance diversity estimates and counteract taxonomic bias in biodiversity assessments. However, the process induces higher costs and time effort and the application in large‐scale biodiversity assessment, e.g., in monitoring schemes needs to be considered on project‐specific prospects. [ABSTRACT FROM AUTHOR]

The efficiency of reduced volume PCR amplification was studied using the VeriFiler™ Express PCR Amplification Kit. Full (25 μL) and reduced (5 μL) volumes were tested in parallel to identify any differences in template DNA sensitivity and other electropherogram parameters. Both volumes produced full DNA profiles down to 0.08 ng/μL DNA concentration at 26 PCR cycles; however, reduced volume produced higher peak heights due to increased signal intensities. Significant difference (p‐value ≤ 0.05) in heterozygote peak height ratios was observed between both volumes, where the reduced volume threshold was lowered to 0.6 to accommodate all data points. However, no significant difference (p‐value > 0.05) was identified in the stutter ratios between both volumes. The analytical threshold for reduced volume was also determined to be 150 RFU with the presence of template DNA in PCR amplification. When the optimized reduced volume parameters were tested on DNA extracted from buccal swab samples using Prep‐n‐Go™ Buffer, good quality DNA profiles were produced. Overall, the reduced volume not only showed better results compared to the full volume, but also enable more samples to be processed with a PCR amplification kit, thus reduced the cost. [ABSTRACT FROM AUTHOR]

Summary: Shigella is the main cause of endemic diarrhoea in low‐income countries. Fast and accurate detection of this pathogen can effectively prevent the consumption of contaminated food and reduce the risk of diarrhoea outbreaks. Recently, a competitive annealing‐mediated isothermal amplification (CAMP) assay was proposed as a novel nucleic acid detection technology. In this study, we developed a visual CAMP assay based on the invasive plasmid antigen H (ipaH) gene for rapid detection of Shigella in milk. Without enrichment culture, the visual CAMP assay could reliably detect Shigella at 2.6 × 102 CFU mL−1 or higher contamination levels. With enrichment culture, even if the spiked level is 1 CFU mL−1, the visual CAMP assay could reliably detect Shigella from the milk sample. Furthermore, a simple paper‐based DNA extraction method was established. The efficiency of the paper‐based method was comparable to that of the commercial kit method, and the entire extraction procedure can be completed in 15 min. The visual CAMP assay combined with the paper‐based DNA extraction method showed higher sensitivity and specificity for Shigella on‐site detection in milk samples. [ABSTRACT FROM AUTHOR]

Forensic entomology aids investigations using insects and is primarily associated with the estimation of post‐mortem interval (PMI). Studies have shown that human DNA can be recovered from the crops of fly larvae. While several factors regarding the recovery of human DNA from crops have been studied, DNA extraction methods have not been thoroughly assessed. Determining a method for optimal extraction could aid crime laboratories in implementing DNA extraction from larvae and streamlining future research. Bovine DNA was used as a substitute for human DNA to test several DNA extractions kits. Four DNA extraction kits (Chelex®, PDQeX forensicGEM, EZ1® DNA Investigator, DNeasy® Powersoil® Pro Kit) were evaluated based on the quantity and quality of bovine DNA extracted. Extractions were performed on whole fly larvae and dissected crops. Quantification was performed using real‐time PCR (qPCR) on a StepOne™ Real‐Time PCR System with SYBR® Green using bovine‐specific cytochrome b primers. The quality of extracts was determined by checking for inhibition using commercial qPCR chemistries with an internal PCR control (IPC). When using whole fly larvae, Powersoil® Pro yielded the highest average DNA yield (n = 10, 0.668 ± 0.458 ng/μl), while EZ1® DNA Investigator yielded the highest average with crops (n = 10, 0.605 ± 0.403 ng/μl). Chelex and forensicGEM yielded low amounts of bovine DNA, and its extracts were inhibited, unlike EZ1® and Powersoil® Pro, which have purification steps. Therefore, it is recommended to use EZ1® DNA Investigator coupled with automation on EZ1® Advanced XL to recover DNA from fly larvae crops. [ABSTRACT FROM AUTHOR]

Bone cells are a suitable substrate for DNA analysis if required to identify the person from whom a sample was taken. Osteocytes, the most abundant cell type in bone, are embedded within mineralized bone matrix. To release DNA from osteocytes for subsequent analyses, either demineralization of the mineral matrix or an overnight incubation is routinely carried out. In this study, we report on a simplified and rapid approach to analyze preserved bone samples that omits this lengthy decalcification process. Nine tibial bone samples were processed to release matrix‐free bone cells after fragmentation without the use of liquid nitrogen. Cell morphology was assessed by microscopy at 220× magnification following staining with Diamond™ Nucleic Acid Dye. Based on the presence of stained nuclei, samples were processed either using a DNA extraction process or by a semi‐direct PCR process. The analysis of the quantity and quality of DNA isolated by both methods was carried out by real‐time PCR and STR profiling to assess inhibition of PCR and DNA degradation. All samples resulted in informative STR profiles with minimal indication of inhibitors. These results demonstrate a potential approach of STR profiling from matrix‐free bone cells within 8 hours without decalcification and DNA extraction. [ABSTRACT FROM AUTHOR]

The present study is a comparative analysis of DNeasy Blood & Tissue Qiagen® kit (Qiagen®, Hilden, Alemanha), salting out, HotShot and phenol–chloroform protocols to extract DNA from sandflies. In addition, a comparative test using sandflies with and without eyes evaluated the potential inhibitory effect in the cPCR. An inhibition test was performed using an exogenous DNA added to the qPCR. The genomic DNA quality of each sample was evaluated by cPCR based on the cytochrome c oxidase subunit I (cox1) gene. The DNA extraction protocols showed the following percentage of amplification: HotShot (91.6% [55/60]), salting out (71.6% [43/60]), phenol–chloroform (95% [57/60]) and kit DNeasy Blood & Tissue Qiagen® (73.3% [44/60]). The phenol–chloroform method achieved a significantly higher frequency of cox1 gene amplification. The pigment present in the phlebotomine's eyes seems to inhibit cPCR reactions since the frequency of amplification of the cox1 gene increased in the sandflies without eyes (p < 0.0001). The HotShot method showed the highest inhibitory potential. These manual extraction techniques can be an inexpensive and effective alternative to study vector–pathogen interactions. [ABSTRACT FROM AUTHOR]

In this study, 10 different traditional Iranian cheeses, which are still consumed by people in rural areas of Iran, were examined to isolate new strains of probiotic bacteria. Isolated bacteria were identified by 16s rRNA gene amplification and subjected to series of in vitro tests to find out their probiotic potential. A total of 2345 colonies were collected and 465 of them were confirmed as lactic acid bacteria (LAB), of which Lactiplantibacillus plantarum, Lactobacillus bulgaricus, and Lacticaseibacillus casei were the top three isolated bacteria. Among the different species of LAB isolated in this study, Lactip. plantarum was the most isolated species, and seven isolates had the significant criteria for being a probiotic strain than other isolates indicating the most adaptable properties of this species. Lactiplantibacillus plantarum was the most resistant bacteria in the bile resistance test and was also the most durable bacteria in gastrointestinal conditions, for example, acidic environment (pH = 2.5) and trypsin. In contrast, Lacticaseibacillus casei was the most susceptible bacterial strain. Lactobacillus rhamnosus showed the most antibacterial effect against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. This study showed that probiotic strains isolated from local cheeses could be considered as suitable biopreservatives and used as specific starter cultures for the production of functional cheeses. [ABSTRACT FROM AUTHOR]

There is an increased use of immunochromatographic test strips to presumptively identify bodily fluids of forensic interest, such as blood, semen, and saliva. Commonly, forensic samples are of low quantities. In the practice of conserving limited samples, it would be ideal to be able to recover the genetic material deposited on these testing membranes. This research aimed to determine whether DNA could be extracted from semen and saliva test strips, which part of the test strip is best to use, and to assess the quality of the DNA recovered. Semen and saliva samples were deposited on SERATEC® PSA Semiquant and Amylase Tests and analyzed. The testing membrane was then removed from the cassette and DNA extraction methods (forensicGEM Universal, forensicGEM Sperm, QIAamp® DNA Mini kit, Monarch® Nucleic Acid Purification kit, and organic extraction) were performed. Quality was evaluated by qPCR and STR analysis. DNA from semen was best extracted using the Monarch® Nucleic Acid Purification kit, while saliva was best extracted using the forensicGEM or QIAamp kits. No significant differences were observed between collection of the sample well pad and testing strip, thus use of the entire strip is encouraged. DNA from semen and saliva was quantifiable with a 1:1000 dilution. DNA quality analysis by qPCR showed that there is no difference in the DNA quality following elution from the test strip. However, degradation was noted in saliva samples and some semen samples by STR analysis. Scientists are encouraged to consider processed test strips for DNA profiling to preserve evidence. [ABSTRACT FROM AUTHOR]

Marine habitats are Earth's largest aquatic ecosystems, yet little is known about marine organism's genomes. Molecular studies can unravel their genetics print, thus shedding light on specie's adaptation and speciation with precise authentication. However, extracting high molecular weight DNA from marine organisms and subsequent DNA library preparation for whole genome sequencing is challenging. The challenges can be explained by excessive metabolites secretion that co‐precipitates with DNA and barricades their sequencing. In this work, we sought to resolve this issue by describing an optimized isolation method and comparing its performance with the most commonly reported protocols or commercial kits: SDS/phenol–chloroform method, Qiagen Genomic Tips kit, Qiagen DNeasy Plant mini kit, a modified protocol of Qiagen DNeasy Plant kit, Qiagen DNeasy Blood and Tissue kit, and Qiagen Qiamp DNA Stool mini kit. Our method proved to work significantly better for different marine species regardless of their shape, consistency, and sample preservation, improving Oxford Nanopore Technologies sequencing yield by 39 folds for Spirobranchus sp. and enabling generation of almost 10 GB data per flow cell/run for Chrysaora sp. and Palaemon sp. samples. [ABSTRACT FROM AUTHOR]

Background and Aims: The characterisation of plant microbiomes using metabarcoding strategies is expected to be progressively replaced by shotgun metagenomic (SMg) approaches. In the present report we explore the potential of applying SMg to grapevine leaf, rhizosphere and soil samples. Methods and Results: Our strategy involved combining a single method for column‐based DNA extraction of multiple tissues, with sequencing using the BGISEQ short‐read next‐generation sequencing platform. This study details aspects of DNA isolation, library construction, sequencing and early bioinformatics treatment of read data, including the use of subsampling as a proxy for detection of sample microbial proportions. The combination of an innovative sequencing platform with recent mapping tools allowed for the characterisation of the microbiome associated with the grapevine leaf, rhizosphere and adjacent soil. Conclusions: We coupled a robust single extraction/library preparation protocol suitable for contrasting samples, with cost‐effective BGISEQ short‐read next‐generation sequencing technology, to generate a landscape of grapevine‐associated microbial diversity. Significance of the Study: Shotgun metagenomics is emerging as a state‐of‐the‐art approach to grapevine microbiome characterisation. The present report provides a detailed and technical roadmap targeting multiple aspects of this important approach. [ABSTRACT FROM AUTHOR]

The root lesion nematode Pratylenchus penetrans parasitizes a wide range of economically important crops, including potato (Solanum tuberosum). Damage by P. penetrans impacts not only the potato yield but can also reduce the tuber quality. Detailed information on tuber infection by P. penetrans is scarce for most cultivars and molecular detection of nematodes from infected tubers is needed. The objective of this study was to assess tuber symptomatology due to P. penetrans infection in 10 potato cultivars and to provide an accurate molecular methodology for nematode detection using tuber peels. Sprouts of certified potato seed from cultivars Agata, Agria, Camel, Désirée, Dirosso, Kennebec, Laura, Picasso, Royata, and Stemster were planted in 2 L pots, and soil was inoculated with 4 P. penetrans/g of soil. Sixty days after inoculation, tubers were harvested, inspected for lesions, and the number of nematodes/g of potato peel assessed. Observations of tubers with symptoms showed the presence of P. penetrans in superficial layers of peels around the lenticels and injured necrotic tissue. Different nematode stages were detected in tubers of all inoculated cultivars, varying from 4 to 46 nematodes/g of potato peel. Species‐specific primers showed suitable sensitivity and reproducibility for the detection of P. penetrans in tuber potato peel samples. The molecular detection of P. penetrans directly from tuber peels can facilitate routine nematode inspections of potato seed tubers or cull potatoes for nematode detection, and prevent further dissemination of this species. [ABSTRACT FROM AUTHOR]

DNA testing of human bones is performed for identification when there is no remaining soft tissue, which often means the samples are old or environmentally compromised. Under these circumstances, it can be difficult to obtain a STR DNA profile. It is important to recover the highest quantity and quality of DNA for STR typing. This study compared the DNA recovery and STR profiles from five anatomical locations in five femora and five tibiae. These locations include the proximal metaphysis, proximal diaphysis, mid‐diaphysis, distal diaphysis, and distal metaphysis. Twenty‐five femur samples and 25 tibia samples were analyzed using the Qiagen Investigator Quantiplex Pro RGQ Kit for quantitating the extracted DNA and the Qiagen Investigator 24plex QS Kit for STR DNA typing. The highest DNA recovery of the five regions tested in both the femur and the tibia was from the midshaft diaphysis. The femur samples resulted in a significantly higher DNA recovery than the tibia samples as analyzed using a Kruskal–Wallis test (P = 0.002103). The midshaft diaphysis and distal diaphysis yielded the most complete STR DNA profiles in the femora, while the distal and proximal diaphysis yielded the most complete STR DNA profiles in the tibiae. There was no correlation between the amount of DNA recovered and the completeness of the STR DNA profile produced with low template extracts in this study. [ABSTRACT FROM AUTHOR]

While several studies have examined temporal DNA degradation in bones collected from terrestrial environments, studies on temporal DNA degradation in bones collected from aquatic environments are limited and mostly based on case studies. The objective of this study was to assess the impact of long‐term submersion, aquatic environment, bone type and DNA extraction method on DNA quality and quantity. Bone samples (scapulae and ribs), collected every ~1000 ADD from a freshwater lake and river, underwent DNA extraction via ChargeSwitch® gDNA Plant Kit and organic phenol–chloroform methods, and DNA quantitation using both TaqMan and SYBR Green‐based quantitative PCR (qPCR) methods. Results suggest that in both bone types, quality of recovered DNA (i.e., degradation index) declined significantly with increase in submersion time. Among two bone types, quality of recovered DNA from scapulae declined faster than rib samples. There was no significant difference in recovered DNA quantity between bone types, DNA extraction methods, or locations but various interactions between these variables showed significant difference. Overall, it can be concluded that DNA can be extracted from waterlogged bone in sufficient quantity to generate an STR profile up to 4000 ADD. [ABSTRACT FROM AUTHOR]

Introduction: Irises have been medicinally used in Ancient Egyptians, Anatolian, Chinese, British and Irish folk medicine. They are also well‐known ornamental plants that have economic value in the perfume industry. The main obvious diagnostic difference between the different species is based on the morphology of the flowers. The flowering cycle is very short as well as the persistence of the fully opened flowers extends for a few days only. Moreover, the climatic conditions significantly causes fluctuation in their blooming time from year to year. This makes the morphological discrimination very difficult. The discrimination of different iris species is of a great importance, as each species is reported to possess different folk medicinal activities. Objectives: Finding genetic and metabolic markers for differentiation between Iris confusa Sealy (Subgen. Limniris Sect. Lophiris), I. pseudacorus L. (Subgen. Limniris Sect. Limniris) and I. germanica L. (Subgen. Iris Sect. Iris) on levels other than traditional taxonomic features. Material and Methods: Inter‐simple sequence repeat (ISSR) and gas chromatography–mass spectrometry (GC–MS) analyses were performed. Results: The highest similarity was found between I. pseudacorus L. and I. germanica L. and the least similarity was between I. confusa Sealy and I. pseudacorus L. The metabolic profiling of the leaves confirmed genetic profiling discriminating I. confusa from the other two species. The primary metabolites of the underground parts showed clear discrimination between the three species. Conclusions: This study represents the sole complete map for distinguishing the three Iris species on genetic and metabolic bases. Inter‐simple sequence repeat and gas chromatography–mass spectrometry (GC–MS) analyses were performed with the aim of finding genetic and metabolic markers for the differentiation between the three Irises (Iris confusa Sealy, I. pseudacorus L. and I. germanica L.) on levels other than their taxonomic characters. The metabolic profiling of the leaves confirmed genetic profiling discriminating I. confusa from the other two species while that of the underground parts showed clear differentiation among the three species. [ABSTRACT FROM AUTHOR]

The extraction of DNA is a fairly standard procedure routinely undertaken in biological academic and research settings. In a forensic DNA context, DNA extraction is the crucial first step toward the generation of DNA profiles used to support convictions or exonerations in the criminal justice system. However, it is often complicated by the additional challenge of yielding sufficient quantities of clean DNA that is free from environmental inhibitors, from a wide spectrum of sample types which may contain limited quantities of biological material. While improvements in methods and technology over the decades have sped up and simplified the process of DNA extraction, several issues remain to be fully addressed—such as the incomplete separation of spermic DNA from sperm‐epithelial cell mixtures, increasing demands for ever‐faster DNA results, or even on‐scene DNA processing, and processing of difficult samples such as skeletal remains or samples contaminated by Chemical, Biological, or Radiological (CBR) agents. These challenges are often compounded by high work volumes and limited quantities of starting material. As such, this review focuses on recent trends and developments in four areas of forensic DNA extraction: (a) analysis of sexual assault evidence which often represents a significant portion of a forensic DNA laboratory's casework, (b) use of portable rapid DNA instruments which has gained much traction among law enforcement in recent times, (c) DNA extraction from difficult and CBR‐contaminated samples which, albeit rare, would be essential in a DNA laboratory's toolbox in the event of an incident, and (d) bypassing DNA extraction altogether. This article is categorized under:Forensic Biology > Forensic DNA Technologies [ABSTRACT FROM AUTHOR]

Premise: Quick and effective DNA extraction from plants for subsequent PCR amplification is sometimes challenging when working across diverse plant taxa that may contain a variety of inhibitory compounds. Time‐consuming methods may be needed to overcome these inhibitory effects as well as the effects of various preservation and collection methods to extract DNA from leaf samples. Our objective was to develop a rapid DNA extraction protocol that could be used with diverse plant taxa to produce high‐quality DNA suitable for downstream PCR applications. Methods and Results: We tested the efficacy of acetone in extracting DNA from fresh, frozen, oven‐dried, acetone‐fixed, and herbarium leaf material of 22 species from 16 woody and herbaceous plant families. An improved simplified DNA extraction protocol was developed using acetone‐fixed leaf material. The addition of 1% sodium dodecyl sulfate solution resulted in the optimal extraction from all tissue samples. The DNA resulting from the extraction protocol was readily amplified using real‐time PCR assays. Conclusions: The protocol described here resulted in the extraction of DNA from recalcitrant plant species that was of sufficient quality and quantity for PCR amplification, as indicated by the low threshold cycle values from real‐time assays. This method is simple, fast, and cost‐effective, and is a reliable tool for extracting high‐quality DNA from plant material containing PCR inhibitors. [ABSTRACT FROM AUTHOR]

Rapid DNA extraction is a critical barrier for routine and fieldable genetics tests for applications in conservation, such as illegal trafficking and fraudulent mislabelling.Here, we develop a non‐thermal focused ultrasound extraction (FUSE) technique that creates a dense cloud of high‐pressure acoustic cavitation bubbles to disintegrate targeted tissues into an acellular debris, resulting in the rapid release of entrapped DNA.In this work, we demonstrate the proof‐of‐concept of the FUSE technique by obtaining species identifiable sequences and shotgun sequencing reads from DNA extracted from Atlantic salmon Salmo salar tissues.Having mitigated the key risks for this technique, we hypothesize future developments with this technology can be applied to accelerate and simplify DNA extraction from exceedingly difficult samples with complex tissue matrices (i.e. fibrous tissue and timber samples) in both laboratory and field settings. [ABSTRACT FROM AUTHOR]

Genomic analysis of hundreds of individuals is increasingly becoming standard in evolutionary and ecological research. Individual‐based sequencing generates large amounts of valuable data from experimental and field studies, while using preserved samples is an invaluable resource for studying biodiversity in remote areas or across time. Yet, small‐bodied individuals or specimens from collections are often of limited use for genomic analyses due to a lack of suitable extraction and library preparation protocols for preserved or small amounts of tissues. Currently, high‐throughput sequencing in zooplankton is mostly restricted to clonal species, that can be maintained in live cultures to obtain sufficient amounts of tissue, or relies on a whole‐genome amplification step that comes with several biases and high costs. Here, we present a workflow for high‐throughput sequencing of single small individuals omitting the need for prior whole‐genome amplification or live cultures. We establish and demonstrate this method using 27 species of the genus Daphnia, aquatic keystone organisms, and validate it with small‐bodied ostracods. Our workflow is applicable to both live and preserved samples at low costs per sample. We first show that a silica‐column based DNA extraction method resulted in the highest DNA yields for nonpreserved samples while a precipitation‐based technique gave the highest yield for ethanol‐preserved samples and provided the longest DNA fragments. We then successfully performed short‐read whole genome sequencing from single Daphnia specimens and ostracods. Moreover, we assembled a draft reference genome from a single Daphnia individual (>50× coverage) highlighting the value of the workflow for non‐model organisms. [ABSTRACT FROM AUTHOR]

The 'One Health' framework emphasizes the ecological relationships between soil, plant, animal and human health. Microbiomes play important roles in these relationships, as they modify the health and performance of the different compartments and influence the transfer of energy, matter and chemicals between them. Standardized methods to characterize microbiomes along food chains are, however, currently lacking. To address this methodological gap, we evaluated the performance of DNA extraction kits and commonly recommended primer pairs targeting different hypervariable regions (V3‐V4, V4, V5‐V6, V5‐V6‐V7) of the 16S rRNA gene, on microbiome samples along a model food chain, including soils, maize roots, cattle rumen, and cattle and human faeces. We also included faeces from gnotobiotic mice colonized with defined bacterial taxa and mock communities to confirm the robustness of our molecular and bioinformatic approaches on these defined low microbial diversity samples. Based on Amplicon Sequence Variants, the primer pair 515F‐806R led to the highest estimates of species richness and diversity in all sample types and offered maximum diversity coverage of reference databases in in silico primer analysis. The influence of the DNA extraction kits was negligible compared to the influence of the choice of primer pairs. Comparing microbiomes using 515F‐806R revealed that soil and root samples have the highest estimates of species richness, while lowest richness was observed in human faeces. Primer pair choice directly influenced the estimation of community changes within and across compartments and may give rise to preferential detection of specific taxa. This work demonstrates why a standardized approach is necessary to analyse microbiomes within and between source compartments along food chains in the context of the One Health framework. [ABSTRACT FROM AUTHOR]

A wet‐vacuum‐based collection method with the M‐Vac® was compared to a wet‐swabbing collection method by examining the recovery of diluted blood on 22 substrates of varying porosity. The wet‐vacuum method yielded more total nuclear DNA than wet‐swabbing on 18 porous substrates, recovering on average 12 times more DNA. However, both methods yielded comparable amounts of total DNA on two porous and two nonporous substrates. In no instance did wet‐swabbing significantly recover more DNA. The wet‐vacuum method also successfully collected additional DNA on previously swabbed substrates. Mitochondrial DNA yields were assessed, and outcomes were generally similar to the nuclear DNA outcomes described above. Results demonstrate that wet‐vacuuming may serve as an alternative collection method to swabbing on difficult porous substrates and could potentially recover additional DNA on previously swabbed substrates. However, swabbing remains the preferred collection method on substrates with visible stains and/or nonporous surfaces for reasons of convenience, simplicity, and lower cost relative to the wet‐vacuum method. [ABSTRACT FROM AUTHOR]

A wet‐vacuum‐based collection method with the M‐Vac® was compared to a wet‐swabbing collection method by examining the recovery of diluted blood on 22 substrates of varying porosity. The wet‐vacuum method yielded more total nuclear DNA than wet‐swabbing on 18 porous substrates, recovering on average 12 times more DNA. However, both methods yielded comparable amounts of total DNA on two porous and two nonporous substrates. In no instance did wet‐swabbing significantly recover more DNA. The wet‐vacuum method also successfully collected additional DNA on previously swabbed substrates. Mitochondrial DNA yields were assessed, and outcomes were generally similar to the nuclear DNA outcomes described above. Results demonstrate that wet‐vacuuming may serve as an alternative collection method to swabbing on difficult porous substrates and could potentially recover additional DNA on previously swabbed substrates. However, swabbing remains the preferred collection method on substrates with visible stains and/or nonporous surfaces for reasons of convenience, simplicity, and lower cost relative to the wet‐vacuum method. [ABSTRACT FROM AUTHOR]

Next‐generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol–chloroform and silica column) to three different sources of DNA (toe pad, skin punch and bone) from 10 historical avian museum specimens. We show that a modified phenol–chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol–chloroform protocol, probably as a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol–chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphism (SNP) markers in anchovy (Engraulis encrasicolus) egg samples were analysed to detect their origin on a small spatial scale (200 km) by assigning genotypes to adult anchovy stocks. The novelty of this work is the application of a rapid high‐throughput method for genotyping each single anchovy egg, in a single execution, using a set of 96 genome‐wide SNPs in a dynamic array system with microfluidic technology (Fluidigm 96.96).The existence of two ecotypes in E. encrasicolus had already been identified based on SNP polymorphism in the Atlantic Ocean and in the Mediterranean Sea, showing that habitat type (offshore versus coastal/estuarine) is the most important component of genetic differentiation among populations of anchovy.In this work, anchovy egg genotypes from areas of the Western Mediterranean were assigned to adult populations. Only two localities in which adult anchovies were sampled represented donor populations for the coastal/estuarine egg genotypes. Although some degree of mixing among the hauls could exist, the assignment of egg groups to adult populations led to distinguishing the contributions of distinct ecotypes to new wild generations. We can conclude that the high rate of egg dispersion caused by marine currents and the different degrees of local retention could explain the genetic heterogeneity observed in the adult populations, where eggs from neighbouring spawning sites tend to mix.The results highlight that this technique represents a new and useful tool for addressing evolutionary questions, breed recognition, assignment, and connectivity assessment of individual eggs, and anchovy population dynamics, for the management of stocks. [ABSTRACT FROM AUTHOR]

Telomere length has been used as a proxy of fitness, aging and lifespan in vertebrates. In the last decade, dozens of articles reporting on telomere dynamics in the fields of ecology and evolution have been published for a wide range of taxa. With this growing interest, it is necessary to ensure the accuracy and reproducibility of telomere length measurement techniques. Real‐time quantitative PCR (qPCR) is routinely applied to measure relative telomere length. However, this technique is highly sensitive to several methodological variables and the optimization of qPCR telomere assays remains highly variable between studies. Therefore, standardized guidelines are required to enable the optimization of robust protocols, and to help in judging the validity of the presented results. This review provides an overview of preanalytical and analytical factors that can lead to qPCR inconsistencies and biases, including: (a) sample type, collection and storage; (b) DNA extraction, storage and quality; (c) qPCR primers, laboratory reagents, and assay conditions; and (d) data analysis. We propose a minimum level of information for publication of qPCR telomere assays in evolutionary ecology considering the methodological pitfalls and sources of error. This review highlights the complexity of the optimization and validation of qPCR for telomere measurement per se, demonstrating the importance of transparency and clarity of reporting methodological details required for reliable, reproducible and comparable qPCR telomere assays. We encourage efforts to implement standardized protocols that ensure the rigour and quality of telomere dynamics studies. [ABSTRACT FROM AUTHOR]

In unison, fingerprinting and DNA analysis have played a pivotal role in forensic investigations. Fingerprint powders that are available on the market can come in a range of colors and with specific properties. This study evaluated the efficiency of DNA extraction from samples coated with 3 brands of fingerprint powders: Lightning, Sirchie, and SupraNano, covering a range of colors and properties. A total of 23 fingerprint powders were tested using the Chelex, Promega DNA IQ™, and Applied Biosystems™ PrepFiler™ DNA extraction protocols. The DNA IQ™ and PrepFiler™ methods extracted higher yields of DNA in comparison to Chelex, which also accounted for better quality of PowerPlex x00AE; 21 DNA profiles recovered. There were no signs of degradation or inhibition in the quantification data, indicating that samples returning low DNA yield was due to interference during DNA extraction and not PCR inhibition. DNA profiles were recovered from the majority of fingerprint powders with only a single powder, Sirchie Magnetic Silver, failing to produce a profile using any of the methods tested. A link was observed between the DNA extraction chemistry, fingerprint powder property, that is, nonmagnetic, magnetic and aqueous, and the brand of fingerprint powder. Overall, the DNA IQ™ method was favorable for nonmagnetic fingerprint powders, while magnetic fingerprint powders produced more DNA profiles when extracted with the PrepFiler™ chemistry. This study highlights the importance of screening DNA extraction chemistries for the type of fingerprint powder used, as there is not a single DNA extraction method that suits all fingerprint powder brands and properties. [ABSTRACT FROM AUTHOR]

Genetic diversity is the determinant for pest species' success and vector competence. Understanding the ecological and evolutionary processes that determine the genetic diversity is fundamental to help identify the spatial scale at which pest populations are best managed. In the present study, we present the first comprehensive analysis of the genetic diversity and evolution of Rhopalosiphum padi, a major pest of cereals and a main vector of the barley yellow dwarf virus (BYDV), in England. We have used a genotyping‐by‐sequencing approach to study whether (a) there is any underlying population genetic structure at a national and regional scale in this pest that can disperse long distances; (b) the populations evolve as a response to environmental change and selective pressures; and (c) the populations comprise anholocyclic lineages. Individual R. padi were collected using the Rothamsted Insect Survey's suction‐trap network at several sites across England between 2004 and 2016 as part of the RIS long‐term nationwide surveillance. Results identified two genetic clusters in England that mostly corresponded to a North–South division, although gene flow is ongoing between the two subpopulations. These genetic clusters do not correspond to different life cycle types, and cyclical parthenogenesis is predominant in England. Results also show that there is dispersal with gene flow across England, although there is a reduction between the northern and southern sites with the south‐western population being the most genetically differentiated. There is no evidence for isolation by distance and other factors such as primary host distribution, uncommon in the south and absent in the south‐west, could influence the dispersal patterns. Finally, results also show no evidence for the evolution of the R. padi population, and it is demographically stable despite the ongoing environmental change. These results are discussed in view of their relevance to pest management and the transmission of BYDV. [ABSTRACT FROM AUTHOR]

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low‐template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re‐extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low‐template DNA sample processing are not enough to justify the risk associated with additional manipulation. [ABSTRACT FROM AUTHOR]

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low‐template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re‐extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low‐template DNA sample processing are not enough to justify the risk associated with additional manipulation. [ABSTRACT FROM AUTHOR]