Microbes from oil reservoirs shape petroleum composition through processes such as biodegradation or souring. Such processes are considered economically detrimental and might pose health and safety hazards. It is therefore crucial to understand the composition of a reservoir\'s microbial community and its metabolic capabilities. However, such analyses are hindered by difficulties in extracting DNA from such complex fluids as crude oil. Here, we present a novel DNA extraction method from oils with a wide American Petroleum Institute (API) gravity (density) range. We investigated the ability to extract cells from oils with different solvents and surfactants, the latter both nonionic and ionic. Furthermore, we evaluated three DNA extraction methods. Overall, the best DNA yields and the highest number of 16S rRNA reads were achieved with isooctane as a solvent, followed by an ionic surfactant treatment using sodium dodecyl sulfate and DNA extraction using the PowerSoil Pro Kit (Qiagen). The final method was then applied to various oils from oil reservoirs collected in aseptic conditions. Despite the expected low cell density of 101-103 cells/ml, the new method yielded reliable results, with average 16S rRNA sequencing reads in the order of 41431 (±8860) per sample. Thermophilic, halophilic, and anaerobic taxa, which are most likely to be indigenous to the oil reservoir, were found in all samples. API gravity and DNA yield, despite the sufficient DNA obtained, did not show a correlation.

With the advent of new molecular diagnostic techniques, retrieving DNA from the formalin-fixed paraffin-embedded (FFPE) tissues has become an essential yet challenging step for efficient downstream processes. Owing to low quality and quantity of DNA retrieved from the FFPE sections, the process is often impractical and needs significant improvements. Here, we established an efficient method for the purification of DNA from FFPE specimens by optimizing incubation temperature, incubation time, and the concentration of a formalin scavenger tris(hydroxymethyl)aminomethane (Tris) for reverse-crosslinking. The optimized method, named \"Highly concentrated Tris-mediated DNA extraction\" (HiTE), yielded three times the DNA yield per tissue slice compared with a representative DNA extraction kit. Moreover, the use of HiTE-extracted DNA increased the yield of the sequencing library three times and accordingly yielded a log higher and more reproducible sequencing library compared with that obtained using the commonly used commercial kit. The sequencing library prepared from HiTE-extracted FFPE-DNA had longer inserts and produced reads that evenly covered the reference genome. Successful application of HiTE-extracted FFPE-DNA for whole-genome and targeted gene panel sequencing indicates its practical usability.

UNASSIGNED: DNA extraction is an important step of any molecular experiment. DNA could not be easily extracted from members of actinomycetes by the usual methods of lysis. Due to the low efficiency of the conventional DNA extraction methods, development of an effective technique for DNA extraction of actinobacteria in emergency cases seems to be necessary. Since most of the DNA extraction techniques and commercial kits are not efficient enough to extract DNA from actinobacteria, this study was conducted to improve an efficient method obtained from conventional one to extract DNA from this group of bacteria.
UNASSIGNED: DNA extraction was performed using five methods (an improved method, Invisorb Spin Plant Mini Kit, EZ-10 Spin Column, Sarrbrucken method (HZI, Germany) and Kirby Bauer\'s method). To evaluate the quantity and quality of extracted genomic DNA, UV absorbance of all samples and efficiency of polymerase chain reaction (PCR) were evaluated.
UNASSIGNED: Overall, the results revealed that the highest quantity (up to 4000 ng/μl) and good quality of DNA was obtained using introduced DNA extraction method.
UNASSIGNED: Results indicated that recently introduced improved method was more efficient for extraction of DNA from actinobacteria for DDH (DNA-DNA hybridization) test and for those require the high concentration of DNA.

BACKGROUND: Liquid biopsy is gaining increasing popularity in cancer screening and diagnosis. However, there is no relatively mature DNA isolation method or commercial kit available that is compatible with different LB sample types. This study developed a PAN-sample DNA isolation method (PAN method) for liquid biopsy samples.
METHODS: The PAN method has two key steps, including biosample-specific pretreatments for various LB sample types and high concentration guanidine thiocyanate buffer for lysis and denaturation procedure. Subsequently, the performance of PAN method was validated by a series of molecular analyses.
RESULTS: The PAN method was used to isolate DNA from multiple sample types related to LB, including plasma, serum, saliva, nasopharyngeal swab, and stool. All purified DNA products showed good quality and high quantity. Comparison of KRAS mutation analysis using DNA purified using PAN method versus QIAamp methods showed similar efficiency. Epstein-Barr virus DNA was detected via Q-PCR using DNA purified from serum, plasma, nasopharyngeal swab, and saliva samples collected from nasopharyngeal carcinoma patients. Similarly, methylation sequencing of swab and saliva samples revealed good coverage of target region and high methylation of HLA-DPB1 gene. Finally, 16S rDNA gene sequencing of saliva, swab, and stool samples successfully defines the relative abundance of microbial communities.
CONCLUSIONS: This study developed and validated a PAN-sample DNA isolation method that can be used for different LB samples, which can be applied to molecular epidemiological research and other areas.

DNA studies of endangered or extinct species often rely on ancient or degraded remains. The majority of ancient DNA (aDNA) extraction protocols focus on skeletal elements, with skin and hair samples rarely explored. Similar to that found in bones and teeth, DNA extracted from historical or ancient skin and fur samples is also extremely fragmented with low endogenous content due to natural degradation processes. Thus, the development of effective DNA extraction methods is required for these materials. Here, we compared the performance of two DNA extraction protocols (commercial and custom laboratory aDNA methods) on hair and skin samples from decades-old museum specimens to Iron Age archaeological material. We found that apart from the impact sample-specific taphonomic and handling history has on the quantity and quality of DNA preservation, skin yielded more endogenous DNA than hair of the samples and protocols tested. While both methods recovered DNA from ancient soft tissue, the laboratory method performed better overall in terms of DNA yield and quality, which was primarily due to the poorer performance of the commercial binding buffer in recovering aDNA.
濒危或已灭绝动物的DNA研究通常依据古代或已严重降解的标本遗存。比较成熟的古DNA（ancient DNA，aDNA）提取方法主要针对于骨骼样本，对于皮张和毛发的研究较为少见。与古代的骨骼和牙齿情况相似，陈旧皮张样品中的DNA也由于自然降解等情况的存在，使得获取到的DNA片段长度极短、内源DNA含量极低。因此，针对这些样品，需要开发有效的DNA提取方法。本研究以几十年前到铁器时代的动物皮张和毛发样品为材料，比较2种提取方法（试剂盒法和古DNA实验室法）及相互组合的2种方法的DNA提取效果。研究发现除样品本身的差别（如年代等），皮张样品比毛发样品保留更多的内源DNA。所有方法都能够从陈旧的皮张样品中获取内源DNA，但是实验室的方法在DNA产量和质量上整体优于其它方法，实验室方法在纯化上的表现优于试剂盒方法。.

Although protocols exist for the recovery of ancient DNA from land snail and marine bivalve shells, marine conch shells have yet to be studied from a paleogenomic perspective. We first present reference assemblies for both a 623.7 Mbp nuclear genome and a 15.4 kbp mitochondrial genome for Strombus pugilis, the West Indian fighting conch. We next detail a method to extract and sequence DNA from conch shells and apply it to conch from Bocas del Toro, Panama across three time periods: recently-eaten and discarded (n = 3), Late Holocene (984-1258 before present [BP]) archaeological midden (n = 5), and mid-Holocene (5711-7187 BP) paleontological fossil coral reef (n = 5). These results are compared to control DNA extracted from live-caught tissue and fresh shells (n = 5). Using high-throughput sequencing, we were able to obtain S. pugilis nuclear sequence reads from shells across all age periods: up to 92.5 thousand filtered reads per sample in live-caught shell material, 4.57 thousand for modern discarded shells, 12.1 thousand reads for archaeological shells, and 114 reads in paleontological shells. We confirmed authenticity of the ancient DNA recovered from the archaeological and paleontological shells based on 5.7× higher average frequency of deamination-driven misincorporations and 15% shorter average read lengths compared to the modern shells. Reads also mapped to the S. pugilis mitochondrial genome for all but the paleontological shells, with consistent ratios of mitochondrial to nuclear mapped reads across sample types. Our methods can be applied to diverse archaeological sites to facilitate reconstructions of the long-term impacts of human behaviour on mollusc evolutionary biology.

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

In this study, strain Streptomyces sp. Act4Zk was isolated based on a method developed for the isolation of myxobacteria. Due to the low efficiency of the majority of conventional DNA extraction techniques, for molecular identification of the strain Streptomyces sp. Act4Zk, a new technique for DNA extraction of Actinobacteria was developed. In order to explore potential bioactivities of the strain, extracts of the fermented broth culture were prepared by an organic solvent (i.e. ethyl acetate) extraction method using. These ethyl acetate extracts were subjected to HPLC fractionation against standard micro-organisms, followed by LC/MS analysis. Based on morphological, physiological, biochemical and 16S rRNA gene sequence data, strain Streptomyces sp. Act4Zk is likely to be a new species of Streptomyces, close to Streptomyces genecies and Streptomyces roseolilacinus. Antimicrobial assay indicated high antifungal activity as well as antibacterial activity against Mycobacterium smegmatis and Gram-positive bacteria for the new strain. HPLC and LC/MS analyses of the extracts led to the identification of three different compounds and confirmed our hypothesis that the interesting species of the genus Streptomyces being a good producer of staurosporine and some derivatives.

Both the host microbiome and the microbiome of the built environment can have profound impacts on human health. While prior studies have suggested that the variability introduced by DNA extraction method is less than typical biologic variation, most studies have focused on 16S rRNA amplicon sequencing or on high biomass fecal samples. Shotgun metagenomic sequencing provides advantages over amplicon sequencing for surveying the microbiome, but is a challenge to perform in lower microbial biomass samples with high human DNA content such as sputum or vacuumed dust. Here we systematically evaluate the impact of four different extraction methods (phenol:choloroform, and three high-throughput kit-based approaches, the Promega Maxwell gDNA, Qiagen MagAttract PowerSoil DNA, and ZymoBIOMICS 96 MagBead). We report the variation in microbial community structure and predicted microbial function assessed by shotgun metagenomics sequencing in human stool, sputum, and vacuumed dust obtained from ongoing cohort studies or clinical trials. The same beadbeating protocol was used for all samples to focus our evaluation on the impact of kit chemistries on sequencing results. DNA yield was overall highest in the phenol:choloroform and Promega approaches. Only the phenol:choloroform approach showed evidence of contamination in negative controls. Bias was evaluated using mock community controls, and was noted across all extraction methods, although Promega exhibited the least amount of bias. The extraction method did not impact the proportion of human reads, although stool had the lowest proportion of human reads (0.1%) as compared to dust (44.1%) and sputum (80%). We calculated Bray-Curtis dissimilarity and Aitchison distances to evaluate the impact of extraction method on microbial community structure by sample type. Extraction method had the lowest impact in stool (extraction method responsible for 3.0-3.9% of the variability), the most impact in vacuumed dust (12-16% of the variability) and intermediate values for sputum (9.2-12% variability). Similar differences were noted when evaluating microbial community function. Our results will inform investigators planning microbiome studies using diverse sample types in large clinical studies. A consistent DNA extraction approach across all sample types is recommended, particularly with lower microbial biomass samples that are more heavily influenced by extraction method.

The skin microbiota is characterized by high intra- and inter-variability among individuals, due to a multitude of intrinsic and extrinsic parameters such as genetics, lifestyles or pollution. This variability may be heightened due to sampling method as the skin is a multilayer organ and its outermost layer consists of dead cells. In order to investigate this biological variability in a reproducible way, we studied how sampling procedure and DNA extraction methods influence the qualitative and quantitative gathering of bacterial communities. Here, we tested a new sampling procedure that consists in exerting a slight abrasion (scrubbing) on the skin prior to swabbing and extracting DNA in order to remove squames and access deeper ecological niches. Scrubbed and non-scrubbed samples were collected from a panel of six volunteers, and four DNA extraction methods were performed on the samples. The abundance, diversity and structure of the bacterial communities were measured using qPCR technics and 16S rDNA gene-metabarcoding. Bacterial community abundance was significantly impacted by the DNA extraction method (in favor of a method designed for tissues) but not by sampling procedure, as scrubbing does not increase bacterial biomass gathered. Bacterial α- and β-diversities were both affected by DNA extraction methods and sampling procedure. Scrubbing reveals different microbial composition by gathering bacteria living in deeper skin layer, resulting in a lower intra-personal variability. The taxonomic analysis showed that more bacteria belonging to anaerobes groups were present in scrubbed samples. We conclude that DNA extraction methods designed for tissue are not necessarily associated with high qualitative efficiency and slight scrubbing prior DNA extraction reduces intrapersonal variability and give access to a new microbial diversity.