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.

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Črnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Črnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations.

Surveying bacterial and archaeal microbial communities in host and environmental studies requires the collection and storage of samples. Many studies are conducted in distant locations challenging these prerequisites. The use of preserving buffers is an important alternative when lacking access to cryopreservation, however, its effectivity for samples with challenging chemistry or samples that provide opportunities for fast bacterial or archaeal growth upon exposure to an aerobic environment, like peat samples, requires methodological assessment. Here, in combination with an identified optimal DNA extraction kit for peat soil samples, we test the application of several commercial and a homemade preservation buffer and make recommendations on the method that can most effectively preserve a microbiome reflective of the original state. In treatments with a non-optimal buffer or in the absence, we observed notable community shifts beginning as early as three days post-preservation lowering diversity and community evenness, with growth-driven artifacts from a few specific phyla. However other buffers retain a very close composition relative to the original state, and we described several metrics to understand some variation across them. Due to the chemical effects of preservation buffers, it is critical to test their compatibility and reliability to preserve the original bacterial and archaeal community in different environments.

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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Significant variation exists in the molecular structure of compact and trabecular bone. In compact bone full dissolution of the bone powder is required to efficiently release the DNA from hydroxyapatite. In trabecular bone where soft tissues are preserved, we assume that full dissolution of the bone powder is not required to release the DNA from collagen. To investigate this issue, research was performed on 45 Second World War diaphysis (compact bone)-epiphysis (trabecular bone) femur pairs, each processed with a full dissolution (FD) and partial dissolution (PD) extraction method. DNA quality and quantity were assessed using qPCR PowerQuant analyses, and autosomal STRs were typed to confirm the authenticity of isolated DNA. Our results support different mechanisms of DNA preservation in compact and trabecular bone because FD method was more efficient than PD method only in compact bone, and no difference in DNA yield was observed in trabecular bone, showing no need for full dissolution of the bone powder when trabecular bone tissue is processed. In addition, a significant difference in DNA yield was observed between compact and trabecular bone when PD was applied, with more DNA extracted from trabecular bone than compact bone. High suitability of trabecular bone processed with PD method is also supported by the similar quantities of DNA isolated by FD method when applied to both compact and trabecular bone. Additionally similar quantities of DNA were isolated when compact bone was extracted with FD method and trabecular bone was extracted with PD method. Processing trabecular bone with PD method in routine identification of skeletonized human remains shortens the extraction procedure and simplifies the grinding process.

A comparative analysis of 26 petrous bones and epiphyses of metacarpals from the Second World War era revealed no significant differences in DNA yield or success in STR typing. This unexpected parity in DNA preservation between the petrous bone, a renowned source of endogenous DNA in skeletal remains, and the epiphyses of metacarpals, which are porous and susceptible to taphonomic changes, is surprising. In this study, we introduced ATR-FTIR spectroscopy as an approach to unravel the correlation between bone molecular structure and DNA preservation. Metacarpals and petrous bones with same taphonomic history were sampled and prepared for DNA analyses. While one portion of the sample was used for DNA analysis, the other underwent ATR-FTIR spectroscopic examination. The normalized spectra and FTIR indices between the epiphyses of metacarpals and petrous bones were compared. Because the taphonomic history of the remains used is relatively short and stable, the ATR-FTIR spectroscopy unveiled subtle structural differences between the two bone types. Petrous bones exhibited higher mineralization, whereas epiphyses contained more organic matter. The unexpected preservation of DNA in the epiphyses of metacarpals can likely be attributed to the presence of soft tissue remnants within the trabeculae. Here observed differences in the molecular structure of bones indicate there are different mechanisms enabling DNA preservation in skeletal tissues.

This study investigates the preservation of DNA in different categories of teeth, including permanent and deciduous, fully developed and not fully developed, in both adults and non-adults. Teeth were sampled from a modern-era cemetery in Ljubljana, Slovenia. DNA extraction was performed using a full demineralisation protocol. DNA quantity and quality were assessed using qPCR analyses, and autosomal STR typing was conducted to verify genetic profiles. Results revealed significant differences in DNA preservation among various tooth categories. Fully developed permanent teeth of adults exhibited the highest DNA yields, attributed to their fully developed roots and thicker cementum, which is rich in DNA. Deciduous teeth, with thinner enamel and cementum, showed lower DNA preservation regardless of developmental stage. Non-adult teeth generally yielded less DNA compared to adults, even when considering only fully developed permanent teeth, indicating factors beyond developmental stage. These findings suggest that, in archaeological and forensic contexts, researchers should prioritize fully developed permanent teeth for DNA analysis due to their superior preservation. Additionally, this study underscores the importance of considering tooth type and developmental stage when selecting samples for genetic analysis in cases where petrous bone is unavailable, expanding our understanding of DNA preservation in human remains.

The equine faecal microbiota is often assessed as a proxy of the microbial community in the distal colon, where the microbiome has been linked to states of health and disease in the horse. However, the microbial community structure may change over time if samples are not adequately preserved. This study stored equine faecal samples from n = 10 horses in four preservation treatments at room temperature for up to 150 h and assessed the resulting impact on microbial diversity and the differential abundance of taxa. Treatments included \"COLD\" (samples packaged with a cool pack), \"CLX\" (2% chlorhexidine digluconate solution), \"NAP\" (nucleic acid preservation buffer), and \"FTA\" (Whatman FTA™ cards). The samples were assessed using 16S rRNA gene sequencing after storage for 0, 24, 72, and 150 h at room temperature under the different treatments. The results showed effective preservation of diversity and community structure with NAP buffer but lower diversity (p = 0.001) and the under-representation of Fibrobacterota in the FTA card samples. The NAP treatment inhibited the overgrowth of bloom taxa that occurred by 72 h at room temperature. The COLD, CLX, and NAP treatments were effective in preserving the faecal microbiota for up to 24 h at room temperature, and the CLX and NAP treatments improved the yield of Patescibacteria and Fibrobacterota in some cases. The cold and CLX treatments were ineffective in preventing community shifts that occurred by 72 h at room temperature. These findings demonstrate the suitability of the COLD, NAP, and CLX treatments for the room temperature storage of equine faeces for up to 24 h and of NAP buffer for up to 150 h prior to processing.

The avian gut microbiota has been the subject of considerable recent attention, with potential implications for diverse fields such as the poultry industry, microbial ecology, and conservation. Faecal microbiotas are frequently used as a non-invasive proxy for the gut microbiota, however the extraction of high-quality microbial DNA from avian faeces has often proven challenging. Here we aimed to evaluate the performance of two DNA preservation methods (95% ethanol and RNAlater) and five extraction approaches (IndiSpin Pathogen Kit, QIAamp PowerFecal Pro DNA Kit, MicroGEM PrepGEM Bacteria Kit, ZymoBIOMICS DNA Miniprep Kit, and an in-house phase separation-based method) for studying the avian gut microbiota. Systematic testing of the efficacy of these approaches on faecal samples from an initial three avian species (chicken, ostrich, and the flightless parrot kākāpō) revealed substantial differences in the quality, quantity and integrity of extracted DNA, but negligible influence of applied method on 16S rRNA gene-based microbiota profiles. Subsequent testing with a selected combination of preservation and extraction method on 10 further phylogenetically and ecologically diverse avian species reiterated the efficacy of the chosen approach, with bacterial community structure clustering strongly by technical replicates for a given avian species. Our finding that marked differences in extraction efficacy do not appear to influence 16S rRNA gene-based bacterial community profiles provides an important foundation for ongoing research on the avian gut microbiota.

DNA extraction and preservation bias is a recurring topic in DNA sequencing-based microbial ecology. The different methodologies can lead to distinct outcomes, which has been demonstrated especially in studies investigating prokaryotic community composition. Eukaryotic microbes are ubiquitous, diverse, and increasingly a subject of investigation in addition to bacteria and archaea. However, little is known about how the choice of DNA preservation and extraction methodology impacts perceived eukaryotic community composition. In this study, we compared the effect of two DNA preservation methods and six DNA extraction methods on the community profiles of both eukaryotes and prokaryotes in phototrophic biofilms on seagrass (Zostera marina) leaves from the Baltic Sea. We found that, whereas both DNA preservation and extraction method caused significant bias in perceived community composition for both eukaryotes and prokaryotes, extraction bias was more pronounced for eukaryotes than for prokaryotes. In particular, soft-bodied and hard-shelled eukaryotes like nematodes and diatoms, respectively, were differentially abundant depending on the extraction method. We conclude that careful consideration of DNA preservation and extraction methodology is crucial to achieving representative community profiles of eukaryotes in marine biofilms and likely all other habitats containing diverse eukaryotic microbial communities.