Quality Of DNA Research Articles

Efficiency comparison of DNA extraction kits for analysing the cockle gut bacteriome

Cockles play a vital ecological role and provide valuable ecosystem services globally. However, the performance, production, and safe consumption of cockles are significantly influenced by their gut-associated bacteriome. Accurate understanding of gut-bacteriome interactions, and surveillance of pathogenic bacteria loads in cockles, rely on efficient DNA extraction methods that yield high-quality and representative bacterial DNA. Despite this importance, reliable extraction methods for cockles are currently overlooked. Therefore, we evaluated the performance of five DNA extraction kits (E.Z.N.A.® Soil DNA; FastDNA® Spin; DNeasy PowerSoil Pro; QIAamp PowerFecal DNA; ZymoBIOMICS™DNA Miniprep) in terms of DNA quality, yield, bacterial community structure (analysed by using denaturating gradient gel electrophoresis; DGGE), and bacteriome composition (analysed by 16S rRNA gene sequencing) in Cerastoderma edule gut. The DNeasy kit provided the highest purity and quantity of bacterial DNA, while the PowerFecal and Zymo kits exhibited reduced extraction efficiency. DGGE profiles revealed significant variability between the tested kits (R = 0.512; mean P = 0.011), but the FastDNA kit under-represented the bacterial community in cockles’ gut. Based on alpha diversity, the DNeasy kit outperformed the others and successfully detected all abundant genera found with the alternative kits. Our findings indicate that the DNeasy kit is an efficient DNA extraction method, enabling a molecular representation of the gut-associated bacteriome in C. edule. These results contribute to the development of effective techniques for studying the cockle gut bacteriome and its ecological implications.

Be bold, start cold! cold formalin fixation of colorectal cancer specimens granted superior DNA and RNA quality for downstream molecular analysis

The use of cold formalin fixation (CFF; i.e., fixating tissue samples with 4 °C precooled formalin) recently attracted further attention owing to its putative improved ability to preserve nucleic acid compared with standard room temperature formalin (SFF). In this study, we aimed to assess the effect of four formalin-based fixation protocols (SFF, CFF, delayed formalin fixation-DFF, and cold formalin hyperfixation; CFH) on both DNA and RNA quality. We collected 97 colorectal cancer (CRC) and analyzed 23 metrics of nucleic acid quantity and quality yield using a multiplatform approach by combining spectrophotometric, fluorimetric, electrophoretic, and polymerase chain reaction (PCR) assays. Following confirmation of fixation-protocol-related different effects via clustering analysis, CFF presented best metrics compared with all protocols, specifically positive coefficients of DV1000-60000, DV2/DV1, DNA λ ratio 260/230, and ABL gene expression absolute copies, and negative coefficient of DV150-1000. The SFF subgroup presented a positive coefficient of DV150-1000 and negative coefficients for DV1000-60000, DV2/DV1, RNA λ ratio 260/230, RNA QuBit concentration, DV100/200, RNA electrophoresis concentration and absolute quantity, and ABL copies. Overall, we confirmed the superior yield performances of CFF preservation for both DNA and RNA compared with the other protocols in our series of CRC samples. Pending further validations and clarification of the specific mechanisms behind these findings, our study supports the implementation of CFF in the pathology unit routine specimen management for tumor tissue molecular profiling.

Genetic Variations in the NRF2 Microsatellite Contribute to the Regulation of Bovine Sperm-Borne Antioxidant Capacity.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor protein-coding gene, considered a master regulator of the cellular stress response. The genetic variations of the NRF2 could influence its transcriptional profile and, subsequently, the stress resilience in all cell types, including sperm cells. Therefore, the sperm-borne antioxidants abundance in association with the genetic variation of a GCC microsatellite located at the 5' upstream region of the NRF2 gene was investigated in young (n = 8) and old (n = 8) Holstein bulls' sperm cells at different seasons. The sperm DNA was sequenced using Sanger sequencing, while- the sperm-borne mRNA analysis was carried out using the synthesized cDNA and qPCR. The data were statistically analyzed using GraphPad Prism 10.0.2 software. The results showed that two bulls had a heterozygous genotype of eight and nine GCC repeats, while biallelic of eight, nine, and fifteen repeats were identified in two, ten, and two bulls, respectively. The computational in silico analysis revealed that the NRF2 upstream sequence with 15, 9, and 8 GCC repeats bound with 725, 709, and 707 DNA-binding transcription factor proteins, respectively. Lower quality of sperm DNA was detected in the spring season compared to other seasons and in young bulls compared to old ones, particularly in the summer and autumn seasons. The mRNA expression analysis revealed that the PRDX1 gene was the abundant transcript among the studied sperm-borne antioxidants and was significantly determined in old bulls' spermatozoa. Moreover, two transcripts of the NRF2 gene and antioxidant (SOD1, CAT, GPX1, TXN1, NQO1) genes displayed differential expression patterns between the age groups across seasons in an antioxidant-dependent manner. The bulls with a heterozygous GCC sequence exhibited elevated sperm-borne mRNA levels of NRF2 and PRDX1 transcripts. Taken together, the findings suggest that the NRF2-GCC microsatellite may contribute to the transcription regulation of NRF2 transcripts and their subsequent downstream antioxidants in bovine sperm cells.

FamLink2 – A comprehensive tool for likelihood computations in pedigrees analyses involving linked DNA markers accounting for genotype uncertainties

There is an increasing demand for software that can handle an arbitrary number of linked markers in forensic genetics; primarily with application to inference of relationships and direct matching but also in applications such as ancestry inference and mixture interpretation. With the emergence of sequencing technologies, denser sets of SNP markers are generated and analyzed. Additionally, sequence data of low quality and quantity DNA generate uncertainty about the underlying true genotype. We provide an efficient implementation of a general model for pedigree likelihood computations with genetic marker data using a three-layered approach. The top and first layer is the population model where allele frequencies and population substructure are accounted for. The second layer is the inheritance model which efficiently handles linked markers using an IBD model. The third and bottom layer is the observational level where we model the likelihood of the true genotype given underlying reads as well as parameters for errors. We exemplify the utility of our implementation as well as provide validation according to guidelines established by the ISFG using a combination of two published SNP panels. We demonstrate that computations are feasible for panels encompassing 10,000 markers and we argue that, due to the properties of the underlying algorithm, extending the number of markers will result in a linear increase in computation time. In addition we study the impact of parameters used in our model and suggest some guidelines pertaining to their values. The results demonstrate that a probabilistic model for low coverage sequence read data is needed instead of relying on an a threshold based genotype and applying our general model for inference of relationships on a real case can be superior, i.e. higher information content, to other methods relying on either fixed genotypes with low quality sequence data or simple pair wise relationship tests. In summary, the implementation, FamLink2 (freely available at https://famlink.se), can jointly handle genetic linkage, genotype uncertainty and population substructure for an arbitrary pedigree with data for any number of individuals. Whereas the current study will focus on calculations disregarding mutations, FamLink2 has the ability to model mutations for certain built-in pedigrees.

Utility of an Archival Dried Blood Spot (DBS) Collection from HIV-Infected Individuals with and without Cancer in a Resource-Limited Setting.

The frequency of virus-associated cancers is growing worldwide, especially in resource-limited settings. One of the biggest challenges in cancer research among people living with HIV (PLWH) has been understanding how infection with both HIV and Kaposi sarcoma-associated herpesvirus (KSHV) promotes the pathogenesis of Kaposi sarcoma (KS), the most common cancer among PLWH worldwide and a significant public health problem in regions with high prevalence of HIV such as Sub-Saharan Africa (SSA). The AIDS and Cancer Specimen Resource (ACSR) provides samples for research, including dried blood spots (DBS) that were collected from large clinical epidemiology studies of KSHV and KS in PLWH conducted more than a decade ago in SSA. Here, we validated the quality of DNA derived from DBS samples from SSA studies and provided evidence of quantitative recovery of inflammatory cytokines using these DBS samples through comparison with paired frozen plasma. Significant differences in DNA, protein yields, and inflammatory biomarker levels were also observed between PLWH with/without KS. Establishing the fitness of DBS samples for studies of KS pathogenesis extends the number of projects that can be supported by these ACSR special collections and provides evidence that DBS collection for future KS research is a practical option in resource-limited settings.

Age-induced changes in skeletal muscle mitochondrial DNA synthesis, quantity, and quality in genetically unique rats

AbstractMitochondrial genomic integrity is a key element of physiological processes and health. Changes in the half-life of the mitochondrial genome are implicated in the generation and accumulation of age-induced mitochondrial DNA (mtDNA) mutations, which are implicated in skeletal muscle aging and sarcopenia. There are conflicting data on the half-life of mtDNA, and there is limited information on how aging affects half-life in skeletal muscle. We hypothesized that skeletal muscle mtDNA synthesis rates would decrease with age in both female and male rats concomitant with changes in mtDNA integrity reflected in mtDNA copy number and mutation frequency. We measured mitochondrial genome half-life using stable isotope labeling over a period of 14 days and assessed mtDNA copy number and deletion mutation frequency using digital PCR in the quadriceps muscle of 9-month-old and 26-month-old male and female OKC-HET rats. We found a significant age-related increase in mtDNA half-life, from 132 days at 9 months to 216 days at 26 months of age in OKC-HET quadriceps. Concomitant with the increase in mtDNA half-life, we found an age-related increase in mtDNA deletion mutation frequency in both male and female rats. Notably, 26-month-old female rats had a lower mutation frequency than male rats, and there were no changes in mtDNA copy number with sex, age, or mitochondrial genotype. These data reveal several key findings: (1) mtDNA turnover in rat skeletal muscle decreases with age, (2) mtDNA half-lives in skeletal muscle are approximately an order of magnitude longer than what is reported for other tissues, and (3) muscle mtDNA turnover differs significantly from the turnover of other mitochondrial macromolecules including components of the mitochondrial nucleoid. These findings provide insight into the factors driving age-induced mtDNA mutation accumulation, which contribute to losses of mitochondrial genomic integrity and may play a role in skeletal muscle dysfunction.

Application of Artificial Intelligence in Sperm Quality Analysis and Sperm Screening

Infertility is a global health issue, and more and more people are hoping to have babies by means of assisted reproductive technology. However, there are still many challenges in fertilization and pregnancy outcomes. Sperm quality is a key factor affecting the success rate of assisted reproduction. Therefore, sperm quality screening is crucial for achieving breakthroughs in assisted reproduction technology. At present, with its capabilities in the field of image recognition, artificial intelligence (AI) is providing new ideas and methods for sperm screening. Various attempts have been made with AI-based models to evaluate indicators such as sperm morphology, DNA quality, and motility level, and some results have been achieved. Herein, we reviewed the application of AI in sperm quality analysis and selection, providing support for the future development of AI and the improvement in the fertilization rate and outcomes of assisted reproductive technology.

The effect of insect excretions/secretions and decomposition fluid on DNA quantity and quality in human bloodstains.

The larval excretions/secretions (ES) of blowflies contain proteolytic enzymes and bacteria that assist with tissue breakdown. Decomposition fluid (DF) contains organic and inorganic waste products from cell death. This study investigated if human DNA recovery from blood was impacted by exposure to ES and DF over time. Lucilia sericata ES were collected daily from 50 larvae, and all available DF was collected from two fetal piglets left to decompose for 2 weeks. Daily for 3-5 days, 28 μL-30 μL of ES, DF, or a 1:1 mixture of the fluids was added to 30 μL of blood on cotton. Three bloodstains per treatment were sampled every 12 h up to 3 days and at 1 and 2 weeks after initial addition of fluid. No PCR inhibition was detected, but DNA degradation increased over time, primarily in samples exposed to ES and ES/DF mixtures. The amount of DNA recovered decreased over time, but generally more DNA was recovered from DF samples than other samples. Full profiles, or partial profiles suitable for routine database searching (14-39 alleles), were generated from all DF and ES samples and at least one mixture sample at all timepoints. Partial profiles of between 1 and 13 alleles were obtained from all other mixture samples, except one mixture sample which generated no profile. These findings indicate bloodstain evidence recovered from maggot-infested and/or decomposing bodies may generate forensically useful DNA evidence and should be analyzed as quickly as possible after collection or stored appropriately to prevent further degradation.

A comprehensive evaluation of Cell Elution Method (CEM) for forensic DNA analysis from smoked cigarettes

Cigarette stubs are commonly encountered trace DNA samples at crime scenes. Standard laboratory practice typically involves direct lysis of the stub for DNA extraction, leading to the co-extraction of DNA-degrading and inhibiting constituents from smoke and tobacco. This process can result in lower-quality DNA profiles. There has been limited focus on developing specific sample processing techniques that minimize these degrading agents and inhibitors before DNA extraction, which could significantly enhance the quality of DNA profiles. This study evaluates a previously established Cell Elution Method (CEM) against the conventional Direct Lysis Method (DLM) for DNA extraction from cigarette stubs. DNA quantity, quality, and subsequent STR profiles were assessed in 80 smoked cigarette stubs, comprising both flavoured and unflavoured types. While CEM exhibited comparable DNA yield from both flavoured (0.17 ng) and unflavoured (0.19 ng) cigarettes, DLM showed significant variability in average DNA yield for unflavoured (0.05 ng) and for flavoured (0.25 ng) cigarettes. Notably, CEM-treated samples demonstrated lower Degradation Index (DI) values compared to DLM-treated ones for both the types of cigarettes. Consequently, STR profiling success rates were higher with CEM, with 95 % of flavoured and 55 % of unflavoured samples yielding informative profiles, compared to 80 % and 0 %, respectively, for DLM. In unflavoured stubs, Amelogenin marker amplification was achieved in 35 % of CEM-treated samples, significantly outperforming the 5 % success rate with DLM. Additionally, CEM resulted in higher average allele recovery rates for both flavoured (58.98 %) and unflavoured (33.41 %) samples compared to DLM. These findings indicate that CEM outperforms DLM in producing higher-quality DNA profiles from cigarette stubs. Thus, CEM can be a choice of method for processing cigarette stub prior to DNA extraction.

Effect of temperature and humidity on insect DNA integrity evaluated by real-time PCR.

Insects collected in dry traps can degrade rapidly, especially in warm, humid environments where many biodiversity and biosecurity surveillance activities are undertaken. Degradation can severely impact diagnostics, as trap catches can become difficult to identify to species level using morphological characters or, of increasing importance, molecular approaches. This is especially problematic for biosecurity surveillance of exotic tephritid fruit flies, where diagnostics are heavily reliant on morphological characters. We tested the effects of differing temperature and humidity conditions on mock samples of tephritid fruit flies in a controlled environment and compared our results to field trap catches. DNA degradation was quantified using real-time PCR assays, including one assay newly developed and tested here. We observed a correlation between increasing DNA degradation and increasing temperature and humidity. The greatest DNA degradation occurred under combined high humidity (90% relative humidity) and constant high temperature (35 °C). Unexpectedly, fluctuating temperature did not have a significant impact on DNA. Other factors, such as trap design, time in the field, and rainfall, did not significantly correlate with DNA quality across the field samples tested. When plotted against mock samples, field samples clustered together, with no clear pattern or predictability regarding the quantity of DNA preserved, indicating other untested environmental variables may be at play. Predictably, increased exposure time was found to have a detrimental effect on DNA quality for all treatments. These findings will improve the delivery of surveillance activities through the implementation of shorter trap clearance timeframes and improved trap designs and procedures.

Comparison of different parts of tangerine (Citrus tangerina) for DNA extraction

The tangerine, a citrus fruit, is grown commercially worldwide in tropical, semitropical, and warm temperate countries, including the Mediterranean region. Tangerine cultivation and production are crucial for the economy of many countries, where genomic DNA plays a critical role in genetic diversity and molecular analysis of this tree. Therefore, this study was conducted in 2022 at Limpopo University to compare DNA extraction from three different parts of the tangerine tree: fresh leaves, stems, and fruit. The Chelex method was used to extract DNA from three samples of C. tangerine from South Africa. The quantity and purity of DNA were measured using a spectrophotometer. PCR amplification with 28S primers was used to confirm and evaluate the extracted DNA. The results from the spectrophotometer showed that the highest quantity of nucleic acid was extracted from the stem (98.18± 12 mg/mL), with a 260/280 ratio for DNA quality (1.85 ± 0.01). The fruit showed a high level of protein (5.8 ± 1.8 mg/mL), but the DNA concentration was lower. In conclusion the amplification of the samples indicated that the stem had high purity and concentration of DNA, which can be suitable for PCR amplification.

Optimal liquid-based DNA preservation for DNA barcoding of field-collected fungal specimens

Preserving fungal tissue DNA in the field is essential for molecular ecological research, enabling the study of fungal biodiversity and community dynamics. This study systematically compares two liquid-based preservation solutions, RNAlater and DESS, for their effectiveness in maintaining macrofungi DNA integrity during field collection and storage. The research encompasses both controlled experiments and real-world field collections. In the controlled experiments, two fungal species were preserved in RNAlater and DESS at different temperatures and durations. DNA extraction success rates were high, but DNA quality and quantity metrics exhibited variations across samples. However, both preservation solutions demonstrated their viability for preserving fungal DNA, with no significant differences between them. In the field-collected macrofungi experiment, 160 paired fungal specimens were preserved in RNAlater and DESS, respectively. Including a drying process to facilitate tissue lysis for DNA extraction significantly impacted the outcomes. RNAlater showed a higher success rate and better DNA quality and quantity compared to DESS. Statistical analysis, including paired and independent t-tests, confirmed significant differences in DNA quality and quantity between the two preservation methods for field-collected samples. This study evaluates RNAlater and DESS for preserving macrofungi DNA in field conditions. Both methods are effective, but RNAlater is superior when a drying step is included in DNA extraction. Researchers can choose based on their specific needs without compromising DNA integrity. These findings advance fungal molecular ecology and DNA preservation strategies in ecological and environmental studies.

Evaluation and Modification of Some Traditional Methods to Extract Genomic DNA in High Quality and Quantity from Different Plant Families

Evaluation and Modification of Some Traditional Methods to Extract Genomic DNA in High Quality and Quantity from Different Plant Families

Implementation of a MSRE ddPCR method for the detection of methylated WIF1 and NPY genes in colorectal cancer patients.

Colorectal cancer is a worldwide leading cause of death accounting for high-rate mortality. Mutations in the epidermal growth factor receptor and RAS/MAPK pathways, as well as altered methylation genes profiles, have been described as molecular mechanisms promoting and sustaining tumour development and progression. Aberrant methylation is a well-known epigenetic mechanism involved in gene regulation; particularly several genes were reported as hypermethylated in CRC. Recently, it was shown that epigenetic alterations in genes such as neuropeptide y, proenkephalin and Wnt inhibitory factor 1 can be used as promising disease biomarkers. Almost all methods developed for the DNA methylation analysis combined next generation sequencing, conventional qRT-PCR or ddPCR with the prior DNA modification with sodium bisulfite. We implemented a ddPCR method to assess the methylation status of Wnt inhibitory factor 1 and neuropeptide y using the methylation sensitive restriction enzyme approach that does not impact on DNA quality and guarantees the discrimination of DNA methylation independent of bisulfite conversion. We showed that this method is robust and sensitive also allowing the monitoring of CRC disease progression when applied to circulating free DNA samples from liquid biopsies, proving to be a fast and easy to implement assay to be used for the monitoring of the methylation pattern of clinically relevant target genes.

Assessment of DNA quality for whole genome library preparation

In recent years, more sophisticated DNA technologies for genotyping have enabled considerable progress in various fields such as clinical genetics, archaeogenetics and forensic genetics. DNA samples previously rejected as too challenging to analyze due to low amounts of degraded DNA can now provide useful information. To increase the chances of success with the new methodologies, it is crucial to know the fragment size of the template DNA molecules, and whether the DNA in a sample is mostly single or double stranded. With this knowledge, an appropriate library preparation method can be chosen, and the DNA shearing parameters of the protocol can be adjusted to the DNA fragment size in the sample. In this study, we first developed and evaluated a user-friendly fluorometry-based protocol for estimation of DNA strandedness. We also evaluated different capillary electrophoresis methods for estimation of DNA fragmentation levels. Next, we applied the developed methodologies to a broad variety of DNA samples processed with different DNA extraction protocols. Our findings show that both the applied DNA extraction method and the sample type affect the DNA strandedness and fragmentation. The established protocols and the gained knowledge will be applicable for future sequencing-based high-density SNP genotyping in various fields.

FecalSeq enrichment with RAD Sequencing from non-invasive environmental samples holds promise for genetic monitoring of an imperiled lagomorph

Despite advances in genomic sequencing and bioinformatics, conservation genomics is still often hindered by a reliance on non-invasive samples. The presence of exogenous DNA and the low quantity and poor quality of DNA in non-invasive samples have been a roadblock to sequencing, thereby limiting the potential for genomic monitoring of endangered species. Recent molecular advances, such as host DNA enrichment, hold promise for facilitating sequencing from non-invasive samples. We used the FecalSeq method to enrich DNA extracted from wild-collected fecal pellets of the imperiled New England cottontail and identified SNPs from 3RAD Sequencing. We obtained SNPs from rabbit pellets, including pellets that were collected in poor environmental conditions and samples that performed poorly with microsatellites. Measures of sequencing success improved with greater amounts of starting DNA and 32% of samples generated SNP genotypes that passed quality control filtering. Genotyping error rates were high, however, and the approach was unable to consistently distinguish unique individuals or matching genotypes, while it was suitable for recovering the expected population structure. Pairing FecalSeq enrichment with RADseq is a promising low-cost method for monitoring wild populations using non-invasive samples in an environmental context, but it may be better suited for informing conservation through population genomics.

A Comparison Study of Cryopreservation of Seminal Fluid between Flinders Technology Associates (FTA-card) and Swab for Multiplex Short Tandem Repeats (STR) Analysis

Background Whatman™ FTA (Flinders Technology Associates) is a form of card soaked in chemicals that denatures proteins while protecting DNA and ensuring the safe handling of dried body fluid spots (blood, semen, and saliva) and buccal cells. To this day, these cards are still infrequently used in forensic science. Therefore, reference samples including biological material may be collected on FTA cards for genetic analysis that have been widely used for DNA preservation and analysis, particularly in forensic sciences and genetic studies. The goal of the study was to evaluate the effects of cryopreservation on DNA quantitative and short tandem repeats (STR) profiling by comparing the yield and quality of DNA extracted before and after cryopreservation using FTA card and swabs in the forensic analysis. The mean total DNA concentration and the purity of seminal fluid from FTA-cards and swabs before and after sperm cryopreservation were measured by using nano-drop spectrophotometer (93.688 ± 12.5) (90.94 ± 13.9) ng/ µl; ( 221.07 ± 20.97)( 135.47 ± 19.6) ng/ µl respectively. Also a highly significant difference (p<0.001) was detected when the results of concentration DNA on FTA card were compared with dilution using wash media before and after sperm cryopreservation. Whereas significant difference (p<0.05) was observed between DNA concentration on FTA card with and without dilution after sperm cryopreservation. STR loci were present in the majority of data, particularly in FTA-card and swab samples where results explained that 100% of STR profiles were present in all 15 loci, both before and after sperm cryopreservation. In contrast to the conventional method of storing and analyzing STR profiling, it can be concluded that seminal fluid samples taken by FTA card and swabs can be taken and stored for a time by cryopreservation, leads to maintaining DNA integration in the field of criminal research.

Refining the Schistosoma haematobium recombinase polymerase amplification (Sh-RPA) assay: moving towards point-of-care use in endemic settings

BackgroundUrogenital schistosomiasis is caused by the parasitic trematode Schistosoma haematobium. Sensitive and specific point-of-care diagnostics are needed for elimination of this disease. Recombinase polymerase amplification (RPA) assays meet these criteria, and an assay to diagnose S. haematobium has been developed (Sh-RPA). However, false-positive results can occur, and optimisation of reaction conditions to mitigate these is needed. Ease of use and compatibility of DNA extraction methods must also be considered.MethodsUsing synthetic DNA, S. haematobium genomic DNA (gDNA), and urine samples from clinical cases, Sh-RPA reactions incorporating different betaine concentrations (0 M, 1 M, 2.5 M, 12.5 M) and the sample-to-water ratios were tested to determine effects on assay specificity and sensitivity. In addition, five commercial DNA extraction kits suitable for use in resource-limited settings were used to obtain gDNA from single S. haematobium eggs and evaluated in terms of DNA quality, quantity, and compatibility with the Sh-RPA assay. All samples were also evaluated by quantitative polymerase chain reaction (qPCR) to confirm DNA acquisition.ResultsThe analytical sensitivity of the Sh-RPA with all betaine concentrations was ≥ 10 copies of the synthetic Dra1 standard and 0.1 pg of S. haematobium gDNA. The addition of betaine improved Sh-RPA assay specificity in all reaction conditions, and the addition of 2.5 M of betaine together with the maximal possible sample volume of 12.7 µl proved to be the optimum reaction conditions. DNA was successfully isolated from a single S. haematobium egg using all five commercial DNA extraction kits, but the Sh-RPA performance of these kits varied, with one proving to be incompatible with RPA reactions.ConclusionsThe addition of 2.5 M of betaine to Sh-RPA reactions improved reaction specificity whilst having no detrimental effect on sensitivity. This increases the robustness of the assay, advancing the feasibility of using the Sh-RPA assay in resource-limited settings. The testing of commercial extraction kits proved that crude, rapid, and simple methods are sufficient for obtaining DNA from single S. haematobium eggs, and that these extracts can be used with Sh-RPA in most cases. However, the observed incompatibility of specific kits with Sh-RPA highlights the need for each stage of a molecular diagnostic platform to be robustly tested prior to implementation.Graphical

Deciphering Trachinotus ovatus resistance to Streptococcus agalactiae:GWAS identification of key SNPs and candidate genes

The genetic basis of resistance in Trachinotus ovatus to Streptococcus agalactiae, a prevalent pathogen in warm-water fish, was explored in this study. T. ovatus, an economically significant marine fish, is widely distributed in tropical and subtropical oceans. This research involved the whole-genome resequencing of 200 T. ovatus individuals and a genome-wide association study (GWAS) to identify significant single nucleotide polymorphisms (SNPs) associated with resistance to S. agalactiae. Methods included infection experiments with S. agalactiae, quality control of DNA samples, high-throughput sequencing, SNP detection and quality control, analysis of population genetic structure, linkage disequilibrium (LD) analysis, GWAS, screening of candidate genes, and genotyping of significant SNPs. Results indicated a significant association of specific SNP loci with resistance in T. ovatus to S. agalactiae, notably SNP18597184 on chromosome LG9, associated with the NTRK3 and IFNK genes involved in neural development, survival, and immune response.

Microbial diversity and gene abundance in denitrifying bioreactors: A comparison of the woodchip surface biofilm versus the interior wood matrix.

Excessive amounts of nitrogen (N) and phosphorus (P) can lead to eutrophication in water sources. Woodchip bioreactors have shown success in removing N from agricultural runoff, but less is known regarding P removal. Woodchip bioreactors are subsurface basins filled with woodchips installed downgradient of agricultural land to collect and treat drainage runoff. Microorganisms use the woodchips as a carbon (C) source to transform N in the runoff, with unresolved biological impacts on P. This study aims to explore microbial communities present in the bioreactor and determine whether milling woodchips to probe the microbial communities within them reveals hidden microbial diversities or potential activities. Metagenomic sequencing and bioinformatic analyses were performed on six woodchip samples (i.e., three unmilled and three milled) collected from a 10-year-old woodchip bioreactor treating agricultural tile drainage. All samples had similar DNA purity, yield, quality, and microbial diversity regardless of milling. However, when sequences were aligned against various protein libraries, our results indicated greater relative abundance of denitrification and P transformation proteins on the outside of the woodchips (unmilled), while the interior of woodchips (milled) exhibited more functional gene abundance for carbohydrate breakdown. Thus, it may be important to characterize microbial communities both within woodchips, and on woodchip surfaces, to gain a more holistic understanding of coupled biogeochemical cycles on N, P, and C in woodchip bioreactors. Based on these findings, we advise that future microbial research on woodchips (and potentially other permeable organic materials) examine both the surface biofilm and the interior organic material during initial studies. Once researchers determine where specific proteins or enzymes of interest are most prevalent, subsequent studies may then focus on either one or both aspects, as needed.