The collection and preservation of biological material before DNA analysis is critical for inter alia biomedical research, medical diagnostics, forensics and biodiversity conservation. In this study, we evaluate an in-house formulated buffer called the Forensic DNA Laboratory-buffer (FDL-buffer) for preservation of biological material for long term at room temperature. Human saliva stored in the buffer for 8 years, human blood stored for 3 years and delicate animal tissues from the jellyfish Pelagia noctiluca comb jelly Beroe sp., stored for 4 and 6 years respectively consistently produced high-quality DNA. FDL-buffer exhibited compatibility with standard organic, salting out and spin-column extraction methods, making it versatile and applicable to a wide range of applications, including automation.
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Octocorals are the most prolific source of terpenoids in the marine environment, with more than 4000 different compounds known from the phylum to date. However, the biochemical and genetic origin of their production remained elusive until recent studies showed that octocorals encode genes responsible for the biosynthesis of terpenoids in their own chromosomal DNA rather than from microbial symbionts as originally proposed. The identified coral genes include those encoding a new group of class I terpene cyclases (TCs) clustered among other candidate classes of tailoring enzymes. Phylogenetic analyses established octocoral TCs as a monophyletic clade, distinct from TCs of plants, bacteria, and other organisms. The newly discovered group of TCs appears to be ubiquitous in octocorals and is evolutionarily ancient. Given the recent discovery of octocoral terpenoid biochemistry and only limited genomic data presently available, there is substantial potential for discovering new biosynthetic pathways from octocorals for terpene production. The following chapter outlines practical experimental procedures for octocoral DNA and RNA extraction, genome and transcriptome assembly and mining, TC cloning and gene expression, protein purification, and in vitro analyses.

Cryptosporidium poses significant public health risks as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized wastewater surveillance methods for Cryptosporidium spp. challenges implementation design and the comparability between studies. Thus, this study compared and contrasted Cryptosporidium wastewater surveillance methods for concentrating wastewater oocysts, extracting oocyst DNA, and detecting Cryptosporidium genetic markers. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome Particles, with and without additional immunomagnetic separation purification (except for the Nanotrap Microbiome Particles). Oocyst DNA extraction by either the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit were evaluated and the impact of bead beating and freeze-thaw pretreatments on DNA recoveries was assessed. Genetic detection via qPCR assays targeting either the Cryptosporidium 18S rRNA gene or the Cryptosporidium oocyst wall protein gene were tested. Oocyst recovery percentages were highest for centrifugation (39-77 %), followed by the Nanotrap Microbiome Particles (24 %), electronegative filtration with a PBST elution (22 %), and Envirocheck HV capsule filtration (13 %). Immunomagnetic separation purification was found to be unsuitable due to interference from the wastewater matrix. Bead-beating pretreatment enhanced DNA recoveries from both the DNeasy Powersoil Pro kit (314 gc/μL DNA) and the QIAamp DNA Mini kit (238 gc/μL DNA). In contrast, freeze-thaw pretreatment reduced DNA recoveries to under 92 gc/μL DNA, likely through DNA degradation. Finally, while both qPCR assays were specific to Cryptosporidium spp., the 18S rRNA assay had a 5-fold lower detection limit and could detect a wider range of Cryptosporidium spp. than the Cryptosporidium oocyst wall protein assay.

In the field of metagenomic research, the choice of DNA extraction methods plays a pivotal yet often underestimated role in shaping the reliability and interpretability of microbial community data. This study delves into the impact of five commercially available DNA extraction kits on the analysis of bovine fecal microbiota. Recognizing the importance of accurate DNA extraction in elucidating microbial community dynamics, we systematically assessed DNA yield, quality, and microbial composition across these kits using 16S rRNA gene sequencing. Notably, the FastDNA spin soil kit yielded the highest DNA concentration, while significant variations in quality were observed across kits. Furthermore, differential abundance analysis revealed kit-specific biases that impacted taxa representation. Microbial richness and diversity were significantly influenced by the choice of extraction kit, with QIAamp DNA stool minikit, QIAamp Power Pro, and DNeasy PowerSoil outperforming the Stool DNA Kit. Principal-coordinate analysis revealed distinct clustering based on DNA isolation procedures, particularly highlighting the unique microbial community composition derived from the Stool DNA Kit. This study also addressed practical implications, demonstrating how kit selection influences the concentration of Gram-positive and Gram-negative bacterial taxa in samples. This research highlights the need for consideration of DNA extraction kits in metagenomic studies, offering valuable insights for researchers striving to advance the precision and depth of microbiota analyses in ruminants.

BACKGROUND: Culex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay.
RESULTS: Genomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55-80%.
CONCLUSIONS: This study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

Kaposi\'s sarcoma (KS) is a cancer affecting skin and internal organs for which the Kaposi\'s sarcoma associated herpesvirus (KSHV) is a necessary cause. Previous work has pursued KS diagnosis by quantifying KSHV DNA in skin biopsies using a point-of-care (POC) device which performs quantitative loop-mediated isothermal amplification (LAMP). These previous studies revealed that extracting DNA from patient biopsies was the rate limiting step in an otherwise rapid process. In this study, a simplified, POC-compatible alkaline DNA extraction, ColdSHOT, was optimized for 0.75 mm human skin punch biopsies. The optimized ColdSHOT extraction consistently produced 40,000+ copies of DNA per 5 µl reaction from 3 mg samples-a yield comparable to standard spin column extractions-within 1 h without significant equipment. The DNA yield was estimated sufficient for KSHV detection from KS-positive patient biopsies, and the LAMP assay was not affected by non-target tissue in the unpurified samples. Furthermore, the yields achieved via ColdSHOT were robust to sample storage in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer prior to DNA extraction, and the DNA sample was stable after extraction. The results presented in this study indicate that the ColdSHOT DNA extraction could be implemented to simplify and accelerate the LAMP-based diagnosis of Kaposi\'s sarcoma using submillimeter biopsy samples.

Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

Multiple methods for collecting genetic samples from amphibians exist, each with their own implications for study design, animal welfare, and costs. Toe clipping is one common method, but there is ongoing debate regarding its potential detriment. Less invasive methods should be implemented, if efficacious, as amphibians are a particularly vulnerable vertebrate group. Skin and buccal swabbing are less invasive methods for genetic sampling, but the potential for contamination and a lower yield of DNA may exist. To compare these methods, we gathered skin swabs, buccal swabs, and toe clips from the same individuals of a relatively small anuran species, Blanchard\'s Cricket Frog (Acris blanchardi). We then compared DNA yield, DNA purity, amplification success rate, and genotypic data quality among sample types. We found toe clips and buccal swabs generated similar DNA yield and purity, with skin swabs yielding significantly less DNA of significantly lower purity than the other sample types. Amplification success rate was significantly higher using toe clips compared to the other sample types, though buccal swab samples amplified more readily than skin swabs. Genotypic data from toe clips and buccal swabs did not differ significantly in quality, but skin swab data quality was significantly lowest among sample types. Thus, skin swabbing could produce erroneous data in some situations, but buccal swabbing is likely an effective substitute to toe clipping, even for small species. Our results can help future researchers select which genetic sampling method might best suit their research needs.

Microbiome studies are becoming larger in size to detect the potentially small effect that environmental factors have on our gut microbiomes, or that the microbiome has on our health. Therefore, fast and reproducible DNA isolation methods are needed to handle thousands of fecal samples. We used the Chemagic 360 chemistry and Magnetic Separation Module I (MSMI) instrument to compare two sample preservatives and four different pre-treatment protocols to find an optimal method for DNA isolation from thousands of fecal samples. The pre-treatments included bead beating, sample handling in tube and plate format, and proteinase K incubation. The optimal method offers a sufficient yield of high-quality DNA without contamination. Three human fecal samples (adult, senior, and infant) with technical replicates were extracted. The extraction included negative controls (OMNIgeneGUT, DNA/RNA shield fluid, and Chemagic Lysis Buffer 1) to detect cross-contamination and ZymoBIOMICS Gut Microbiome Standard as a positive control to mimic the human gut microbiome and assess sensitivity of the extraction method. All samples were extracted using Chemagic DNA Stool 200 H96 kit (PerkinElmer, Finland). The samples were collected in two preservatives, OMNIgeneGUT and DNA/RNA shield fluid. DNA quantity was measured using Qubit-fluorometer, DNA purity and quality using gel electrophoresis, and taxonomic signatures with 16S rRNA gene-based sequencing with V3V4 and V4 regions. Bead beating increased bacterial diversity. The largest increase was detected in gram-positive genera Blautia, Bifidobacterium, and Ruminococcus. Preservatives showed minor differences in bacterial abundances. The profiles between the V3V4 and V4 regions differed considerably with lower diversity samples. Negative controls showed signs from genera abundant in fecal samples. Technical replicates of the Gut Standard and stool samples showed low variation. The selected isolation protocol included recommended steps from manufacturer as well as bead beating. Bead beating was found to be necessary to detect hard-to-lyse bacteria. The protocol was reproducible in terms of DNA yield among different stool replicates and the ZymoBIOMICS Gut Microbiome Standard. The MSM1 instrument and pre-treatment in a 96-format offered the possibility of automation and handling of large sample collections. Both preservatives were feasible in terms of sample handling and had low variation in taxonomic signatures. The 16S rRNA target region had a high impact on the composition of the bacterial profile.
OBJECTIVE: Next-generation sequencing (NGS) is a widely used method for determining the composition of the gut microbiota. Due to the differences in the gut microbiota composition between individuals, microbiome studies have expanded into large population studies to maximize detection of small effects on microbe-host interactions. Thus, the demand for a rapid and reliable microbial profiling is continuously increasing, making the optimization of high-throughput 96-format DNA extraction integral for NGS-based downstream applications. However, experimental protocols are prone to bias and errors from sample collection and storage, to DNA extraction, primer selection and sequencing, and bioinformatics analyses. Methodological bias can contribute to differences in microbiome profiles, causing variability across studies and laboratories using different protocols. To improve consistency and confidence of the measurements, the standardization of microbiome analysis methods has been recognized in many fields.

UNASSIGNED: Symbiotic relationships with diverse microorganisms are crucial for many aspects of insect biology. However, while our understanding of insect taxonomic diversity and the distribution of insect species in natural communities is limited, we know much less about their microbiota. In the era of rapid biodiversity declines, as researchers increasingly turn towards DNA-based monitoring, developing and broadly implementing approaches for high-throughput and cost-effective characterization of both insect and insect-associated microbial diversity is essential. We need to verify whether approaches such as high-throughput barcoding, a powerful tool for identifying wild insects, would permit subsequent microbiota reconstruction in these specimens.
UNASSIGNED: High-throughput barcoding (\"megabarcoding\") methods often rely on non-destructive approaches for obtaining template DNA for PCR amplification by leaching DNA out of insect specimens using alkaline buffers such as HotSHOT. This study investigated the impact of HotSHOT on microbial abundance estimates and the reconstructed bacterial community profiles. We addressed this question by comparing quantitative 16S rRNA amplicon sequencing data for HotSHOT-treated or untreated specimens of 16 insect species representing six orders and selected based on the expectation of limited variation among individuals.
UNASSIGNED: We find that in 13 species, the treatment significantly reduced microbial abundance estimates, corresponding to an estimated 15-fold decrease in amplifiable 16S rRNA template on average. On the other hand, HotSHOT pre-treatment had a limited effect on microbial community composition. The reconstructed presence of abundant bacteria with known significant effects was not affected. On the other hand, we observed changes in the presence of low-abundance microbes, those close to the reliable detection threshold. Alpha and beta diversity analyses showed compositional differences in only a few species.
UNASSIGNED: Our results indicate that HotSHOT pre-treated specimens remain suitable for microbial community composition reconstruction, even if abundance may be hard to estimate. These results indicate that we can cost-effectively combine barcoding with the study of microbiota across wild insect communities. Thus, the voucher specimens obtained using megabarcoding studies targeted at characterizing insect communities can be used for microbiome characterizations. This can substantially aid in speeding up the accumulation of knowledge on the microbiomes of abundant and hyperdiverse insect species.