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Sequencing can provide genomic characterisation of a specific organism, as well as of a whole environmental or clinical sample. High Throughput Sequencing (HTS) makes it possible to generate an enormous amount of genomic data at gradually decreasing costs and almost in real-time. HTS is used, among others, in medicine, veterinary medicine, microbiology, virology and epidemiology. The paper presents practical aspects of the HTS technology. It describes generations of sequencing, which vary in throughput, read length, accuracy and costs ̶ and thus are used for different applications. The stages of HTS, as well as their purposes and pitfalls, are presented: extraction of the genetic material, library preparation, sequencing and data processing. For success of the whole process, all stages need to follow strict quality control measurements. Choosing the right sequencing platform, proper sample and library preparation procedures, as well as adequate bioinformatic tools are crucial for high quality results.

application/pdf, Whilst molecular biological articles have been extensively described in high school-level textbook guidelined by MEXT, it is, however, commonly deemed to be practically difficult to perform molecular biological experiments in high schools. We herein report an ingenious protocol for molecular biological experiments best suitable for biology classes in Japanese high schools, comprising DNA extraction, DNA detection, PCR and agarose electrophoresis, all of which can be completed substantially within no more than two classes. In order to exploit the protocol, we cautiously cared the following points: sets of the experiments can be accomplished in typical period in high schools (50 minutes); and most of the listed experiments can be performed in standard high school facilities. For DNA extraction, “silica monolith” spin column (Animos Co. Ltd.) was employed, much facilitating and shortening the procedure (completed within ~10 minutes). DNA from rice powder (O. sativa) and flour (T. aestivum) was successfully detected by means of agarose electrophoresis, followed by PCR amplification by Taq polymerase reaction mixture (Quick Taq® HS DyeMix; Toyobo Co. Ltd.), targeted for RuBisCO small subunit gene through O. sativa specific primers. The amplification was confirmed by agarose electrophoresis, non-hazardously stained by Midori Green (Nippon Genetics Co. Ltd.). To further develop the experimental protocol, it is thought to be necessary not only to supply the equipment required for routine molecular biological experiments in the high schools, but also to manufacture either illustrated or visualised protocol, expecting for future facilitation of molecular biological learning in high schools.

Marine sponges have recently been recognized as natural samplers of environmental DNA (eDNA) due to their effective water filtration and their ubiquitous, sessile, and regenerative nature. However, laboratory workflows for metabarcoding of sponge tissue have not been optimized to ensure that these natural samplers achieve their full potential for community survey. We used a phased approach to investigate the influence of DNA isolation procedures on the biodiversity information recovered from sponges. In Phase 1, we compared three treatments of residual ethanol preservative in sponge tissue alongside five DNA extraction protocols. The results of Phase 1 informed which ethanol treatment and DNA extraction protocol should be used in Phase 2, where we assessed the effect of starting tissue mass on extraction success and whether homogenization of sponge tissue is required. Phase 1 results indicated that ethanol preservative may contain unique and/or additional biodiversity information to that present in sponge tissue, but blotting tissue dry generally recovered more taxa and generated more sequence reads from the wild sponge species. Tissue extraction protocols performed best in terms of DNA concentration, taxon richness, and proportional read counts, but the non-commercial tissue protocol was selected for Phase 2 due to cost-efficiency and greater recovery of target taxa. In Phase 2 overall, we found that homogenization may not be required for sponge tissue and more starting material does not necessarily improve taxon detection. These results combined provide an optimized DNA isolation procedure for sponges to enhance marine biodiversity assessment using natural sampler DNA metabarcoding., This study was funded by the UK Natural Environment Research Council grant NE/T007028/1. A.R. was also supported by the Spanish Ministry of Science and Innovation grant (RYC2018-024247-I) and CSIC intramural grant (202030E006). M.B.A. was supported by the National Agency for Research and Development (ANID), fellowship program Postdoctorado en el extranjero/2019-74200143.

Telomere length (TL) has emerged as a promising replicative cellular aging marker that reflects both genetic and non-genetic influences. Quantitative PCR (qPCR) TL measurement has been favored as a cost-effective method that can be easily implemented, especially in population studies with limited quantities of source material. However, several recent reports have revealed inconsistencies in telomere length measurements when applying different DNA extraction methods to the same source material. In this study we tested three DNA extraction methods on saliva samples from 48 participants of the National Growth and Health Study (NGHS) collected with DNA Genotek’s Oragene kit. The chosen extraction kits represent three distinct approaches to genomic DNA extraction from lysed cells and we employed two different operators to carry out all assays on the same samples. We measured DNA yield and quality and calculated the between-operator agreement of qPCR TL measurements (intraclass correlation, ICC). Our analyses showed that while both QIAamp and Agencourt DNAdvance had higher agreement between the 2 operators (ICC=0.937, CI [0.891, 0.965] and ICC=0.95, CI [0.911, 0.972] respectively), compared to PrepIT kit (ICC=0.809, CI [0.678, 0.889]), QIAamp extracted DNA samples were notably degraded. Using generalizability theory, we found that the participant-by-extraction-method interaction explained about 10% of total variation in TL, suggesting that TL differences across methods are somewhat participant-specific. Therefore, our results suggest that the among the three DNA extraction methods tested, Agencourt (magnetic bead purification) is the preferred kit, and we also strongly recommend against combining different extraction methods within a study population.

BackgroundEnvironmental water samples are increasingly recognised as an important reservoir of antimicrobial resistance (AMR) genes. Polymerase chain reaction (PCR) and next generation sequencing (NGS) offer a potentially inclusive surveillance platform for a wide range of AMR genes. However, molecular methods are dependent upon the extraction of DNA of high yield and quality. Current options for DNA extraction from complex environmental matrices for downstream molecular applications are either expensive or low yielding. We present here a novel magnetic bead-based DNA extraction method, for the detection of antimicrobial resistance genes (ARGs) from river water in Malawi, named MagnaExtract. MethodsMagnaExtract involves initial filtration of 250ml freshwater, followed by an overnight incubation of the filter in 15ml buffered peptone water (BPW), common procedure in microbiology laboratories. 200{micro}l is then taken for a boil (95{degrees}C) and spin step and mixed with magnetic beads to bind DNA. Following washes with ethanol, the DNA is eluted in nuclease-free water. To determine the effectiveness of this method, 98 freshwater samples were collected from two rivers in Southern Malawi, and DNA was isolated using the MagnaExtract method, two commercial Qiagen (Germany) kits; PowerWater and DNeasy Blood and tissue, alongside a boil and spin of BPW, and a boil and spin from bacterial isolate grown on agar media. All samples were screened with a high-resolution melt (HRM) PCR panel previously validated for the detection of third generation cephalosporin and carbapenem ARGs. We compared the DNA yield obtained using all extraction methods, as well as the identification of each ARG. ResultsDNA yield using MagnaExtract was statistically greater than both boil and spin methods and DNeasy Blood & Tissue (Qiagen, Germany). DNA yield was slightly lower than using PowerWater (Qiagen) but the difference was not statistically significant. MagnaExtract was the only method to identify ARGs in all 98 water samples compared with PowerWater (n=82), DNeasy (n=95) boilate of BPW (n=75) and boilate of bacterial isolate (n=87). The most commonly detected ARG was OXA-48 (n=93). In addition, we found overnight incubation in non-selective enrichment broth (BPW) to promote the growth of bacteria harbouring extended spectrum beta lactamase (ESBL) genes and reduction in the detection of carbapenemase genes. ConclusionThe MagnaExtract approach offers a simple, affordable, high yielding DNA extraction method for the detection of ARGs isolated from river water samples.

Formalin-fixed, paraffin embedded (FFPE) tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research and clinical applications. However, FFPE DNA extraction presents unique challenges and requires lengthy incubation periods, which can be impractical for certain applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE generates a dense bubble cloud of acoustic cavitation capable of ablating tissue into an acellular lysate. FUSE treatment was applied to de-paraffinized porcine pancreas FFPE scrolls, followed by heated incubation for formaldehyde-induced DNA-protein crosslink reversal. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity ratios compared to conventional methods. DNA extracted via FUSE showed comparable fragmentation to conventional methods, and three out of four samples successfully amplified via PCR, indicating suitability for downstream analysis. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications. Master of Science Formalin-fixed, paraffin embedding (FFPE) has historically been the most popular method of biological tissue preservation, as it allows tissue to remain shelf stable for decades. As such, FFPE tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE treatment applies rapid, focused ultrasound waves to tissue, resulting in the mechanical breakdown of cells and subsequent release of DNA. FUSE treatment was applied to pig pancreatic FFPE samples. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity compared to conventional methods. Three out of four DNA samples extracted via FUSE were successfully amplified, and DNA fragment lengths were comparable between FUSE and conventional methods, showing that FUSE did not fragment DNA beyond useful fragment lengths. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications.

A nitrocellulose–graphene oxide hybrid that consists of a commercially nitrocellulose (NC) membrane non-covalently modified with graphene oxide (GO) microparticles was successfully prepared for oligonucleotide extraction. The modification of NC membrane was confirmed by Fourier Transform Infrared Spectroscopy (FTIR), which highlighted the principal absorption bands of both the NC membrane at 1641, 1276, and 835 cm−1 (NO2) and of GO in the range of 3450 cm−1 (CH2-OH). The SEM analysis underlined the well-dispersed and uniform coverage of NC membrane with GO, which displayed thin spider web morphology. The wettability assay indicated that the NC–GO hybrid membrane exhibited slightly lower hydrophilic behavior, with a water contact angle of 26.7°, compared to the 15° contact angle of the NC control membrane. The NC–GO hybrid membranes were used to separate oligonucleotides that had fewer than 50 nucleotides (nt) from complex solutions. The features of the NC–GO hybrid membranes were tested for extraction periods of 30, 45, and 60 min in three different complex solutions, i.e., an aqueous medium, an α-Minimum Essential Medium (αMEM), and an αMEM supplemented with fetal bovine serum (FBS). The oligonucleotides were desorbed from the surface of the NC–GO hybrid membrane using Tris-HCl buffer with a pH of 8.0. Out of the three media utilized, the best results were achieved after 60 min incubation of the NC–GO membranes in αMEM, as evidenced by the highest fluorescence emission of 294 relative fluorescence units (r.f.u.). This value corresponded to the extraction of approximately 330–370 pg (≈7%) of the total oligo-DNA. This method is an efficient and effortless way to purify short oligonucleotides from complex solutions.

Techniques for the identification human and non-human biological samples are developing at very high rates with the advent of different DNA extraction methods and polymerase chain reaction (PCR) based assays. This study aimed at using different extraction protocols to determine the integrity of DNA samples extracted from bovine bone samples of different ages collected from abattoir in Ikorodu, Lagos State, Nigeria. The DNA was extracted using CTAB, PCI protocols and a DNA kit (Quick DNA MiniPrep Plus Kit). Bovine mtDNA fragment containing the gene encoding ATPase 8 was amplified via Polymerase Chain Reaction (PCR). The PCR products were analysed on 1.8% agarose gel. It was observed that the DNA samples extracted using the PCI method at 48 h incubation time had the highest purity (1.68) and concentration (336 ng/µl) compared to other extraction methods employed in the study. However, DNA kit extracted samples had mean ± SE purity (1.52 ± 0-05) and concentration (192.25 ± 31.41 ng/µl) values that were higher than CTAB protocol values but lower than PCI protocol values. All isolated DNA samples were PCR-worthy and thus yielded PCR products which were within the expected amplicon size of 126-bp. All DNA extraction protocols employed in this study are stable and efficient for use in the identification of aged non- human bones. This study also revealed that these protocols can be used to isolate PCR amplifiable DNA from old bones.

AbstractSexual assault samples often contain mixtures of cells coming from at least two donors. Ideally, one would need to separate the cells into two cellular fractions: one consisting of the alleged aggressor’s spermatozoa (the sperm fraction) and the other containing the victim’s epithelial cells (the non-sperm fraction). This separation increases the probability of obtaining the alleged offender’s autosomal DNA profile. However, spermatozoa are often collected along with an excess of biological material originating from the victim, and with unfavorable male:female biological material ratios, the absence of separation could result in the PCR amplification of the victim’s DNA profile only. Several approaches are available to enrich/purify the spermatozoa present on sexual assault samples. In this paper, we compare a new method, the MACSprep™ Forensic Sperm MicroBead Kit (MACSprep, based on microbeads conjugated with antibodies bound to spermatozoa and their retention within a magnetic column) with the Erase Sperm Isolation Kit (Erase, a standard differential lysis separation procedure combined with a specific removal of free DNA) routinely used in our lab. The performance of both kits was tested using sets of vaginal and buccal swabs loaded with different dilutions of sperm, or azoospermic semen, representing a total of 120 independent samples. For the samples containing undiluted sperm, an average recovery of 58% was observed for the MACSprep’s sperm fractions and 43% for Erase’s. Significantly better recovery of azoospermic semen was observed in MACSprep’s non-sperm fractions (~ 85%) compared to Erase (~ 28%). Erase performed significantly better than MACSprep in terms of recovery for diluted sperm samples (1:10 to 1:800 sperm dilutions) in the presence of vaginal cells, while the purities of the achieved sperm fractions were in favor of MACSprep for the highest sperm dilutions tested. Similar trends were observed with buccal swabs loaded with 1:200 sperm dilutions. Increased sperm dilutions on vaginal swabs resulted in higher variability in the male material recovered, whatever the separation method used. Both methods were easy to perform and resulted in male DNA extracts ready to use in less than 2 h. Both kits showed their specificities in terms of recovery efficiency and purity of the sperm fractions. Ideally, additional experiments should be performed in different laboratories, using workflow and chemistries different than ours, to better define the peculiarities observed with MACSprep for high dilutions. Improving the recovery of MACSprep for diluted samples, in addition to its better purity observed in the experiments performed, could make it a method of choice for laboratory workflow, despite MACSprep’s current price per sample being about twice the price of Erase’s.

Considerable skill is required to identify copepods at the species level based on their morphological characteristics. However, DNA analysis does not require advanced microscopy techniques and provides objective data on the phylogenetic relationships between samples. Therefore, DNA analysis is useful as an alternative method for taxonomic studies of copepods. The lysis buffer method by Lee & Frost (2002) is a simple protocol for extracting DNA from single copepod samples. In this method, a fixative, such as formalin, is first replaced with ethanol and a buffer solution. Then, the copepod sample is lysed in the lysis buffer containing a proteolytic enzyme. Thus far, we have conducted DNA extraction of single copepod samples using this method and performed gene amplification by PCR. However, due to the low success rate of PCR amplification, genetic data could not be obtained for approximately 50% of the formalin-fixed samples. In this study, we improved the lysis buffer method with the aim of enhancing the success rate of DNA extraction and PCR amplification from single copepod samples. In addition, the effect of formalin fixation time on PCR amplification was also examined.Zooplankton samples were collected from Manazuru Port, Sagami Bay on September 14, 2017, using a plankton net with a mesh size of 180 μm and fixed with 5% neutralized formalin-seawater. Adult females of the calanoid copepod Acartia japonica were selected from these samples and stored individually in ethanol. DNA was extracted from these copepods via a modified ethanol removal method, with adjustments made to the dilution of the lysis buffer, and incubation time. The mitochondrial cytochrome b gene was amplified from these DNA samples by PCR. When the concentration of the PCR product was 20 ng μL-1 or more, PCR amplification was considered to be successful. Based on the conditions optimized by the above studies, the effect of the formalin fixation time on the PCR amplification of copepods was also investigated. A higher success rate was obtained when natural drying or vacuum drying was performed to remove ethanol during DNA extraction rather than removal by pipetting. Since there was no significant difference between the results of natural drying and vacuum drying, natural drying, which is easier to perform, was selected as the optimum method. We also confirmed that a high success rate was maintained without diluting the lysis buffer after the inactivation of proteolytic enzyme. Regarding the incubation time for lysis, changing from the conventional 60 minutes to 30 minutes did not result in a significant decrease in the success rate of PCR amplification. Thus, the success rate of PCR increased to approximately 90%. Additionally, compared with the conventional lysis buffer method, the number of steps was reduced by half, and the required time was shortened from 1.5 hours to approximately 50 minutes. Furthermore, we confirmed that this improved lysis buffer method can be applied to single cells of small protozoa such as flagellates and ciliates. The effect of formalin fixation time on PCR amplification of the mitochondrial cytochrome b gene after DNA extraction via this improved lysis buffer method was investigated. As expected, the success rate of PCR amplification decreased with the formalin fixation time. However, when the fixation period was within 1 month, PCR products with a concentration of more than 20 ng μL-1 were obtained in 95% of the individual copepod samples. Furthermore, even after 3 months, similar concentrations of PCR product were obtained in 80% of individuals.Genetic analysis of small zooplankton is increasingly important not only in taxonomy but also for biodiversity and phylogeographic studies. The data presented in this study will be very important and useful in such studies.

Iridoviruses are DNA virus and have caused huge economic losses in the aquaculture industry. The aim of this study was to establish a colorimetric loop-mediated isothermal amplification (LAMP) protocol for the on-site detection of Singapore grouper iridovirus (SGIV). The SGIV-VP61 gene was chosen as the target gene to develop a colorimetric LAMP assay. The optimized condition of the colorimetric LAMP assay was incubation at 63 °C for 1 h. Samples infected with SGIV could be detected with the color change from yellow into pink. The sensitivity of the developed assay is 5.66 copies/μL of the viral DNA template. This sensitivity was about 1000 times higher than that of conventional PCR while it was slightly lower than the one-step semi-nested PCR assay. A total of 60 DNA samples extracted from the fin tissue of the SGIV-infected Asian seabass were examined for SGIV by colorimetric LAMP, semi-nested PCR and conventional PCR. The results of the colorimetric LAMP assay showed 94.87 % agreement with the semi-nested PCR. In addition, the DNA extraction method using NaOH showed a better performance in the colorimetric LAMP assay. Taken together, the colorimetric LAMP established was a sensitive, rapid and specific method for the detection of SGIV. SGIV was not detected in samples randomly taken from a genetically improved line of the Asian seabass. However, some seabass obtained from the local markets were found to contain SGIV. Thus, the LAMP assay has the potential application in the diagnosis of iridovirus diseases in the aquaculture industry.

Extracting DNA from degraded human remains poses a challenge for any forensic genetics laboratory, as it requires efficient high-throughput methods. While little research has compared different techniques, silica in suspension has been identified in the literature as the best method for recovering small fragments, which are often present in these types of samples. In this study, we tested five DNA extraction protocols on 25 different degraded skeletal remains. Including the humerus, ulna, tibia, femur, and petrous bone. The five protocols were organic extraction by phenol/chloroform/isoamyl alcohol, silica in suspension, High Pure Nucleic Acid Large Volume silica columns (Roche), InnoXtract™ Bone (InnoGenomics), and PrepFiler™ BTA with AutoMate™ Express robot (ThermoFisher). We analysed five DNA quantification parameters (small human target quantity, large human target quantity, human male target quantity, degradation index, and internal PCR control threshold), and five DNA profile parameters (number of alleles with peak height higher than analytic and stochastic threshold, average relative fluorescence units (RFU), heterozygous balance, and number of reportable loci) were analysed. Our results suggest that organic extraction by phenol/chloroform/ isoamyl alcohol was the best performing method in terms of both quantification and DNA profile results. However, Roche silica columns were found to be the most efficient method.

Background PacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing. Results We developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively. Conclusions DNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.

Analysis of several parameters is required for adequate quality control in umbilical cord blood units (UCBU) when used for therapeutic purposes.To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU.One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the sequence-specific priming technique.CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p0.0001), as well as that of UCB in comparison with that of the segment (p0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY/11 and GP5/GP6+.Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos.Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU.Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming.La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p0.0001); así como la de SCU comparada con la de segmento (p0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+.Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

The human milk microbiota (HMM) of healthy women can vary substantially, as demonstrated by recent advances in DNA sequencing technology. However, the method used to extract genomic DNA (gDNA) from these samples may impact the observed variations and potentially bias the microbiological reconstruction. Therefore, it is important to use a DNA extraction method that is able to effectively isolate gDNA from a diverse range of microorganisms. In this study, we improved and compared a DNA extraction method for gDNA isolation from human milk (HM) samples to commercial and standard protocols. We evaluated the extracted gDNA using spectrophotometric measurements, gel electrophoresis, and PCR amplifications to assess its quantity, quality, and amplifiability. Additionally, we tested the improved method’s ability to isolate amplifiable gDNA from fungi, Gram-positive and Gram-negative bacteria to validate its potential for reconstructing microbiological profiles. The improved DNA extraction method resulted in a higher quality and quantity of the extracted gDNA compared to the commercial and standard protocols and allowed for polymerase chain reaction (PCR) amplification of the V3–V4 regions of the 16S ribosomal gene in all the samples and the ITS-1 region of the fungal 18S ribosomal gene in 95% of the samples. These results suggest that the improved DNA extraction method demonstrates better performance for gDNA extraction from complex samples such as HM.

Wastewater monitoring became a promising solution in the early detection of outbreaks. Despite the achievements in the identification of pathogens in wastewater using real-time PCR, there is still a lack of reliable rapid nucleic acid extraction protocols. Therefore, in this study, samples were subjected to alkali, proteinase K and/or bead-beating followed by reverse purification magnetic beads-based separation. Wastewater samples spiked with S. aureus, E. coli and C. parvum were used as examples for Gram-positive and -negative bacteria and protozoa, respectively. All results were compared with a spin column technology as a reference method. Proteinase K with bead beating (vortexing with 0.1 mm glass beads for three minutes) was particularly successful for bacterial DNA extraction (three- to five-fold increase). The most useful extraction protocol for protozoa was pre-treatment with proteinase K (eight-fold increase). The selected methods were sensitive as far as detecting one bacterial cell per reaction for S. aureus, ten bacterial cells for E. coli and two oocysts for C. parvum. The extraction reagents are cold chain independent and no centrifuge or other large laboratory equipment is required to perform DNA extraction. A controlled validation trial is needed to test the effectiveness at field levels.

The Earth’s atmosphere plays a critical role in transporting and dispersing biological aerosols. Nevertheless, the amount of microbial biomass in suspension in the air is so low that it is extremely difficult to monitor the changes over time in these communities. Real-time genomic studies can provide a sensitive and rapid method for monitoring changes in the composition of bioaerosols. However, the low abundance of deoxyribose nucleic acid (DNA) and proteins in the atmosphere, which is of the order of the contamination produced by operators and instruments, poses a challenge for the sampling process and the analyte extraction. In this study, we designed an optimized, portable, closed bioaerosol sampler based on membrane filters using commercial off-the-shelf components, demonstrating its end-to-end operation. This sampler can operate autonomously outdoors for a prolonged time, capturing ambient bioaerosols and avoiding user contamination. We first performed a comparative analysis in a controlled environment to select the optimal active membrane filter based on its ability to capture and extract DNA.We have designed a bioaerosol chamber for this purpose and tested three commercial DNA extraction kits. The bioaerosol sampler was tested outdoors in a representative environment and run for 24 h at 150 L/min. Our methodology suggests that a 0.22- m polyether sulfone (PES) membrane filter can recover up to 4 ng of DNA in this period, sufficient for genomic applications. This system, along with the robust extraction protocol, can be automated for continuous environmental monitoring to gain insights into the time evolution of microbial communities within the air., Internal funding to Pump-prime Interdisciplinary Research and Impact Activities’ from the University of Aberdeen granted to T.M. J.B.R., QUADRAT NERC Doctoral Training Partnership, UKRI. M.-P.Z. was supported by grant PID2019- 104205GB-C21 funded by MCIN/AEI/10.13039/501100011033.

By offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long-read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long-read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short-read sequencing. The challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long-read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol-chloroform (PC) extraction and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions, as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read-size distribution, assembly performance and the number of circularized elements found in sequencing results suggest that column-based kits with enzyme supplementation, rather than PC methods, may be more appropriate for long-read sequencing of metagenomes.

Palm oil production is related to the number of fruit and is thought to be determined since the beginning of the flowering phase as the initial stage of fruit formation. Identification of oil palm flowing genes needs to be done as the first step of knowing the mechanism of flowering in oil palm molecular. Oil male and palm females contain high polysaccharides and polyphenolic compounds so they can inhibit the process of molecular identification. Molecular studies provide accurate and fast information about the potential of oil palm genetics as a commercial commercial commodity. The purity and concentration of DNA and the temperature of annealing are the main requirements for PCR-based molecular studies. The purpose of this study obtained the extraction method of oil palm flowers which produced high DNA concentration and purity and optimized the temperature of annealing to identify the flowering gene. DNA from male flower spike and female flowers is extracted using the A (SDS) method, method B (CTAB), method C (CTAB + PVP), and method D (commercial kit). Primary BMS annealing temperature for amplification of optimized flowering genes using PCR gradients. The extraction of male flowers and female flowers using the C (CTAB + PVP) method produces the best DNA concentration and purity compared to other methods. The best annealing temperature for male flower DNA amplification and female flowers using the BMS primer is 61.1 o C. Amplification of male flowers and female flowers using Primary BMS produces 1200 PB products. The results of DNA extraction and PCR amplification using the BMS primers in female flower samples are better than male flowers

The analysis of trace DNA is a crucial component in forensic applications. Biological materials containing low-level DNA collected at crime scenes, such as fingerprints, can be valuable as evidence. Automatic detection of biological samples has been largely embraced in forensic applications to meet the increasing throughput requirements. However, the amount of DNA automatically retrieved from trace evidence often tends to be small and unstable, ultimately resulting in poor detection of DNA profiles. Thus, in this work, we introduced a robust DNA extraction and purification platform named Bionewtech® BN3200 (Bionewtech®, Shanghai, China) with the goal of constructing a rapid automatic detection system for trace DNA. The establishment of automatic detection system for trace DNA mainly encompassed two parts: assessing the sensitivity of automatic extraction platform and screening the optimal short tandem repeat (STR) typing kit. The sensitivity of Bionewtech® BN3200 platform based on Ultra-sensitive DNA Extraction kit was initially estimated, demonstrating that this extraction platform might contain large potential in the trace DNA extraction. For the amplification part, three sets of commercial multiplex STR typing kits were selected as candidates, and the amplified products were further genotyped on the Applied Biosystems 3500xl Genetic Analyzer. After comparation, SiFa™ 23 Plex Kit was determined as the most suitable amplification system for trace DNA. Eventually, the newly exploited trace DNA detection system was successfully implemented in the detection of fingerprints derived from glass surfaces with the five-seconds contact time. As a result, the DNA recovered from the fingerprints fluctuated approximately from 57.60 pg to 18.05 ng, in addition, over 70% of the total STR loci were detected in 75% of the fingerprint samples.

Oak wilt caused by Bretziella fagacearum is an important disease of Quercus spp.; however, its diagnosis may be confused with damage resulting from other diseases, insects, or abiotic factors. Laboratory diagnosis is important in such situations and when disease control action is desired. PCR tests can provide accurate lab diagnosis within 2 days. Two variations of a simple DNA extraction protocol using sodium hydroxide (NaOH) were compared with that of the proprietary protocol of a commercially available kit (CK) for nested PCR to detect the pathogen in oak sapwood. High frequencies of pathogen detection (98 to 100% of 48 branch segments assayed) were found for northern pin oak using the two NaOH-based and the CK methods. Detection rates were similar but lower for bur oak (ranged from 58 to 79%) and white oak (ranged from 54 to 71%) regardless of DNA extraction method. Using our alternative DNA extraction protocols may reduce total time and cost of B. fagacearum detection in PCR-based diagnosis and other downstream applications.

Objective Autogenous Staphylococcus pseudintermedius bacterins can reduce prescribing of antimicrobials in the management of canine recurrent pyoderma. However, increasing prevalence of meticillin-resistant, mecA-positive S. pseudintermedius (MRSP) raises concern over dispersal of mecA through bacterin therapy. We investigated the presence and integrity of mecA in bacterin formulations after manufacturing. Material and methods Twenty clinical isolates (12 MRSP, 7 MR-S. aureus, 1 meticillin-susceptible SP) were investigated. Pellets from overnight growth were washed 3 times with 0.5 % phenol saline, followed by addition of 0.1 ml 10 % formal-saline to 10 ml phenol-saline. Sterility was confirmed, and DNA extracted using both a standard genomic extraction kit and one recommended for formalin-fixed tissue samples (FFPE). The presence of mecA was determined after PCR and its integrity examined in 5 randomly selected samples after sequencing. Results In all bacterins from meticillin-resistant isolates, mecA was detected following FFPE extraction; products aligned fully to a reported mecA sequence. After standard DNA extraction, mecA was seen in 16/19 samples. Conclusion Persistence of mecA in MRSP bacterins suggests that dispersal of this important resistance mediator through therapy may be possible. While the ability of skin bacteria to uptake naked DNA remains unclear, it seems prudent to only formulate autogenous bacterins from mecA-negative S. pseudintermedius to avoid unnecessary spread of mecA.

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections at a relatively low cost, within a limited timeframe, to support global collaborations.

Cronobacter sakazakii is a foodborne, emerging opportunistic pathogen that causes severe bacteremia, necrotizing enterocolitis, and sepsis with a mortality rate of up to 80%. In this study, we developed a simple and sensitive fluorescent turn-off aptasensor with rolling circle amplification assay for viable C. sakazakii detection in powdered infant formula. The results showed that the proposed aptasensor has good performance and specificity for detecting viable C. sakazakii in pure culture and powdered infant formula samples within 3 h. Under the optimal reaction conditions, there is a linear relationship between fluorescent intensity at 490 nm and logarithmic concentration of C. sakazakii in the range of 2.7 × 105 to 2.7 × 102 cfu/mL, with a limit of detection of 2.7 × 102 cfu/mL in pure culture. The proposed aptasensor achieved a recovery of 104 to 111% in pure culture, and 96 to 107% in spiked powdered infant formula samples. The proposed aptasensor does not require complicated DNA extraction steps or antibodies, and can be performed at 37°C, making it a convenient and sensitive strategy for C. sakazakii detection.

Leptospirosis diagnosis by MAT requires antibody levels that are typically present only after the first week of symptoms, many days after infection. To improve testing capacity and to develop a fast and reliable solution for the diagnosis of this disease in the first few days after clinical manifestations, the National Reference Laboratory for Leptospirosis/WHO Collaborating Center in Brazil implemented a duplex molecular method by qPCR for human samples for the detection of the gene lipL32, conserved in pathogenic Leptospira spp. In this paper, we describe the overall performance of this protocol in the first 3 months as a standard routine. Detection of pathogenic Leptospira spp. DNA was similar between blood, plasma, and tissue samples, with a limit of detection as low as one cell per sample, and among 391 samples from suspected cases, 174 (44.6%) were positive. The average RNASEP1 control gene detection cycle thresholds (Ct) were 28.4 and 29.8 for positive and negative samples, respectively. The median sample collection interval from the beginning of symptoms was 3 days for positive and 4 days for negative samples, respectively. Neither age, sex, nor the time intervals between sample collection and DNA extraction significantly influenced the results. Surprisingly, positivity was related to the time between DNA extraction and the qPCR reaction. These data support the use of this routine as a diagnostic approach to strengthen the molecular detection of leptospirosis and to develop new strategies.

This thesis investigates the problem of how to sample terrestrial environments for bioprospecting for pharmaceutically useful bacterial secondary metabolites. There is already a considerable literature on the ecology of bacteria in terrestrial ecosystems as well as on attempts to bioprospect from particular terrestrial environments. The first chapter reviews both of these kinds of studies and outlines unresolved questions that need to be answered to efficiently sample for bioprospecting. Most studies of bacterial communities in soil use molecular methods and start by extracting DNA by bead beating. The extracted DNA is then PCR amplified using 16S bacterial primers. Bead beating is usually done with the Mobio kit, however, there is no one standard 16S primer pair that is used in most studies. The second chapter tests how comparible results from these studies are. DNA was extracted from soil and compost using the Mobio kit and a contrasting method and amplified with four different primer pairs. Results showed that primers have more effect on results than extraction method but that the Mobio kit underextracts several genera by orders of magnitude. Most antibiotics from bacteria that are in use come from the Actinobacterial phylum. The third chapter uses several primer pairs for Sanger sequencing and bacterial community fingerprinting to compare the Actinobacterial community structure of a range of environments under different Anthropogenic impact. It found that street sediments were enriched in Actinomycetes compared to soil. In the fourth chapter, environmental DNA from soil and street sediments was amplified with primers for type I polyketide synthase (PKSI) genes. Amplicons were sequenced with Sanger sequencing. Several PKSI amplicons were common to soil or street sediment from 1000s of km apart. Soil PKSI sequences were more often non Actinomycetal and more novel than street PKSI sequences.

Extracting high-molecular-weight DNA is critical for successful long-read DNA sequencing. Here, we present a DNA preparation procedure combining the CTAB DNA extraction protocol and a short-read eliminator kit, which consistently produces high yields of high-molecular-weight DNA in the freshwater microcrustacean Daphnia. This method only requires common chemicals and equipment, providing researchers a viable alternative to DNA extraction kits that often require specialized tools.

Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since its introduction for the neonatal screening of phenylketonuria almost 50 years ago. From that time onwards, the range of its applications has been extended until today, including next-generation sequencing (NGS) for molecular genetics diagnosis. This study aimed to evaluate a standardized organic method to extract DNA from DBS samples, for its use in the diagnostic setting.Methods: The clinical applicability of the method was tested using three samples collected in a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from three 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and its performance was assessed through standard PCR, Restriction Length Polymorphism, Sanger sequencing, and Targeted Next-generation sequencing (TNGS).Results: Results were compared with the ones obtained with DNA samples extracted following the internally validated in-house extraction protocol that used six 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to presenting a lower cost per sample., Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since its introduction for the neonatal screening of phenylketonuria almost 50 years ago. From that time onwards, the range of its applications has been extended until today, including next-generation sequencing (NGS) for molecular genetics diagnosis. This study aimed to evaluate a standardized organic method to extract DNA from DBS samples, for its use in the diagnostic setting. Methods: The clinical applicability of the method was tested using three samples collected in a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from three 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and its performance was assessed through standard PCR, Restriction Length Polymorphism, Sanger sequencing, and Targeted Next-generation sequencing (TNGS). Results: Results were compared with the ones obtained with DNA samples extracted following the internally validated in-house extraction protocol that used six 3-mm punches of DBS and samples extracted from whole blood. Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to presenting a lower cost per sample.

Background and aims – Keraunea is a Brazilian endemic genus that has sat uncomfortably in Convolvulaceae where it was placed due to an enlarged and adnate fruit bract typical of Neuropeltis. A recent molecular phylogeny suggested that two of its five morphologically almost identical species actually belong to two different families, Malpighiaceae (superrosids) and Ehretiaceae (superasterids). Later studies have demonstrated that Keraunea effectively belongs to Ehretiaceae, but the proposal of one species belonging to Malpighiaceae has remained problematic. In this study, we re-assess this hypothesis, discuss the issues that have led to this assumption, and offer insights on the importance of carefully using herbarium collections and incorporating morphological evidence in systematic studies. Material and methods – Sequences of matK, rbcL, and ITS for all 77 currently accepted genera of Malpighiaceae, K. brasiliensis and Elatinaceae (outgroup) were compiled from GenBank and analysed with Maximum Likelihood and Bayesian Inference criteria for nuclear, plastid and combined datasets. Additional database and herbarium studies were performed to locate and analyse all duplicates of the holotype of K. brasiliensis to check for misidentified or contaminated material. Key results – Our examination of expanded DNA datasets and herbarium sheets of all K. brasiliensis isotypes revealed that a mistake in tissue sampling was, in fact, what led to this species being proposed to belong in Malpighiaceae. Kew’s isotype had a leaf of Malpighiaceae (likely Mascagnia cordifolia) stored in the fragment capsule, which was sampled and sequenced instead of the actual leaves of K. brasiliensis. Recently published studies have settled the placement of Keraunea in Ehretiaceae (Boraginales) and proposed three additional species. Conclusions – DNA sequences can be helpful in classifying taxa when morphology is conflicting or of a doubtful interpretation, with molecular phylogenetic placement being established as a popular tool accelerating the discovery of systematic relationships. Nonetheless, molecular techniques are also susceptible to methodological mistakes, which necessitates building a solid foundation of plant morphology and taxonomy to avoid artefacts in phylogenetic studies.

Aquatic emergent insect communities form an important link between aquatic and terrestrial ecosystems, yet studying them is costly and time-consuming as they are usually diverse and superabundant. Metabarcoding is a valuable tool to investigate arthropod community compositions, however high-throughput applications, such as for biomonitoring, require cost-effective and user-friendly procedures. To investigate if the time-consuming and labour-intensive DNA purification step can be omitted in metabarcoding, we studied the difference in detection rates and individual read abundance using standard DNA purification versus direct PCR protocols. Metabarcoding with and without DNA purification was performed with artificially created communities of known composition as well as on natural communities both of the dipteran family Chironomidae to compare detection rates, individual read abundances and presence-absence community composition. We found that the here presented novel approach of direct PCR metabarcoding did not alter detection rates and had a minor effect on individual read abundances in artificially created communities. Furthermore, presence-absence community compositions of natural chironomid communities were highly comparable using both approaches. In conclusion, we showed that direct PCR protocols can be applied in chironomid metabarcoding approaches, with possible application for a wider range of arthropod taxa, enabling us to study communities more efficiently in the future.

Pancreatic cancer is highly aggressive and at the time of diagnosis, 80% of patients are in the metastatic stage. Personalize d therapy guided by genomic biomarkers for pancreatic cancer has only recently begun to be investigated.This study aimed to comprehensively identify possible activating and pathological mutations in metastatic pancreatic cancer with NGS of liquid biopsies and to investigate miRNAs and mutations as therapeutic targets as we evaluated their relationships and effect on prognoses Seventeen patients and 20 healthy volunteers were included in the study. Blood samples were taken from the patients, cell free DNA was isolated from the serum, and mutation profiles were analyzed by NGS. Serum miRNA levels were analyzed by isolating circulating miRNA from the same samples from all groups. In patients with G288G synonymous mutants in the HNF1A gene compared to non-mutant patients, miR-17, miR-24, and miR-150 levels were found to be statistically significantly lower;The miR-17 and miR-146a levels of patients with a TP53 gene Pro72Arg mutation were found to be statistically significantly higher than that of the non-mutant group Survival was lower with the c.2472 C>T mutation located in exon 18 of the PDGFRA gene; higher in patients with P72R mutations in the TP53 gene and those with miRNA 17 and 146 upregulation carrying this mutation. This study is clinically meaningful in revealing possible pathogenic mutations detected in tumor cells circulating in metastatic pancreatic cancer, both in terms of prognostic value and whether the information can be used to target treatment, including gene therapy. © 2023, Yuzuncu Yil Universitesi Tip Fakultesi. All rights reserved.

Bacterial abundance data from MiSeq Illumina, Shell script for preprocessing of raw sequences and R scripts for data filtering and analyses were deposited in Cirad data repository (https://dataverse.cirad.fr) and are available from this link: https://doi.org/10.18167/DVN1/D31UAV.; International audience; 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.

There are different variables that affect the success of Touch DNA recovery, including surface type, the collection method used and extraction techniques. This experiment investigated how a range of porous and non-porous surfaces, different DNA collection (cotton swab, nylon flocked swab and SceneSafe Fast™ minitape) and extraction methods (PrepFiler Express BTA™ and QIAamp® DNA Investigator) affected touch DNA recovery.

The quality and quantity of the extracted DNA are two key aspects for a successful PCR (Polymerase Chain Reaction) amplification. Moreover, reduction in time and cost required for DNA extraction are also two considerable factors, in cases when large number of samples are to be analyzed within a limited time-frame and budget. Accordingly, the aim of this study was to compare and optimize performance of five different DNA extraction methods by boiling meat tissues from cattle, buffalo, sheep, goat, chicken, camel, horse and dog in PBS (Phosphate Buffer Saline), distilled water, alkaline lysis buffers 1, 2 or 3. The results indicated that the boiling of meat and its products in alkaline lysis buffers was a good method to extract crude DNA. The optimized crude DNA extraction protocol was coupled with PCR-RFLP (Restriction Fragment Length Polymorphism) analysis for meat species differentiation. This developed workflow was tested on fifty-three commercial beef and mutton samples, out of which three samples were found to be adulterated. In conclusion, the rapid crude DNA extraction protocol has led to the development of a direct PCR-RFLP workflow that is simple, time-saving and cost-effective for PCR-based identification of different meat species.

Production profiling technology is an important method for monitoring the dynamics of oil and gas reservoirs which can effectively improve the efficiency of oil recovery. Production profiling is a technique in which a test instrument is lowered from the tubing to the bottom of the well to measure flow, temperature, pressure, and density in a multi-layer section of a producing well. Normal production profiling process needs to stop production, operate complex, consume long time and high cost. Furthermore, the profile cannot be continuously monitored for a long time. To address these limitations, this paper proposes a production profiling interpretation method based on reservoir primitive microbial DNA sequencing. The microbial stratigraphic baseline with high-resolution features is obtained by sampling and DNA sequencing of produced fluid and cuttings from different wells. Specifically, the random forest algorithm is preferred and improved by comparing the accuracy, precision, recall, F1-score, and running time of three clustering methods: Naïve-Bayes classifier, random forest classifier, and back-propagation classifier. Constructing PSO-random forest model is based on stratigraphic records and produced fluid bacteria features. The computational accuracy and efficiency of this method allows it to describe the production profile for each formation. Moreover, this test process does not need to stop production with simple operation and does not pollute the formation. Meanwhile, by sampling fluid production at different stages, it can achieve the purpose of long-term effective dynamic monitoring of the reservoir.

Fungal species participate in vast numbers of processes in the landscape around us. However, their often cryptic growth, inside various substrates and in highly diverse species assemblages, has been a major obstacle to thorough analysis of fungal communities, hampering exhaustive description of the fungal kingdom. Recent technological developments allowing rapid, high-throughput sequencing of mixed communities from many samples at once are currently having a tremendous impact in fungal community ecology. Universal DNA extraction followed by amplification and sequencing of fungal species-level barcodes such as the nuclear internal transcribed spacer (ITS) region now enable identification and relative quantification of fungal community members across well-replicated experimental settings. Here, we present the sample preparation procedure presently used in our laboratory for fungal community analysis by high-throughput sequencing of amplified ITS2 markers. We focus on the procedure optimized for studies of total fungal communities in humus-rich soils, wood, and litter. However, this procedure can be applied to other sample types and markers. We focus on the laboratory-based part of sample preparation, that is, the procedure from the point where samples enter the laboratory until amplicons are submitted for sequencing. Our procedure comprises four main parts: (1) universal DNA extraction, (2) optimization of PCR conditions, (3) production of tagged ITS amplicons, and (4) preparation of the multiplexed amplicon mix to be sequenced. The presented procedure is independent of the specific high-throughput sequencing technology used, which makes it highly versatile.

Research on the relationship between changes in the gut microbiota and human disease, including AIDS, is a growing field. However, studies on the eukaryotic component of the intestinal microbiota have just begun and have not yet been conducted in HIV-infected patients. Moreover, eukaryotic community profiling is influenced by the use of different methodologies at each step of culture-independent techniques. Herein, initially, four DNA extraction protocols were compared to test the efficiency of each method in recovering eukaryotic DNA from fecal samples. Our results revealed that recovering eukaryotic components from fecal samples differs significantly among DNA extraction methods. Subsequently, the composition of the intestinal eukaryotic microbiota was evaluated in HIV-infected patients and healthy volunteers through clone sequencing, high-throughput sequencing of nuclear ribosomal internal transcribed spacers 1 (ITS1) and 2 (ITS2) amplicons and real-time PCRs. Our results revealed that not only richness (Chao-1 index) and alpha diversity (Shannon diversity) differ between HIV-infected patients and healthy volunteers, depending on the molecular strategy used, but also the global eukaryotic community composition, with little overlapping taxa found between techniques. Moreover, our results based on cloning libraries and ITS1/ITS2 metabarcoding sequencing showed significant differences in fungal composition between HIV-infected patients and healthy volunteers, but without distinct clusters separating the two groups. Malassezia restricta was significantly more prevalent in fecal samples of HIV-infected patients, according to cloning libraries, whereas operational taxonomic units (OTUs) belonging to Candida albicans and Candida tropicalis were significantly more abundant in fecal samples of HIV-infected patients compared to healthy subjects in both ITS subregions. Finally, real-time PCR showed the presence of Microsporidia, Giardia lamblia, Blastocystis and Hymenolepis diminuta in different proportions in fecal samples from HIV patients as compared to healthy individuals. Our work revealed that the use of different sequencing approaches can impact the perceived eukaryotic diversity results of the human gut. We also provide a more comprehensive view of the eukaryotic community in the gut of HIV-infected patients through the complementarity of the different molecular techniques used. Combining these various methodologies may provide a gold standard for a more complete characterization of the eukaryotic microbiome in future studies.

Immune cell-type composition changes with age, potentially weakening the response to infectious diseases. Profiling epigenetics marks of immune cells can help us understand the relationship with disease severity. We therefore leveraged a targeted DNA methylation method to study the differences in a cohort of pneumonia patients (both COVID-19 positive and negative) and unaffected individuals from peripheral blood. This approach allowed us to predict the pneumonia diagnosis with high accuracy (AUC = 0.92), and the PCR positivity to the SARS-CoV-2 viral genome with moderate, albeit lower, accuracy (AUC = 0.77). We were also able to predict the severity of pneumonia (PORT score) with an R2 = 0.69. By estimating immune cellular frequency from DNA methylation data, patients under the age of 65 positive to the SARS-CoV-2 genome (as revealed by PCR) showed an increase in T cells, and specifically in CD8+ cells, compared to the negative control group. Conversely, we observed a decreased frequency of neutrophils in the positive compared to the negative group. No significant difference was found in patients over the age of 65. The results suggest that this DNA methylation-based approach can be used as a cost-effective and clinically useful biomarker platform for predicting pneumonias and their severity. © 2022 Informa UK Limited, trading as Taylor & Francis Group.

The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.

Backgrounds PCR is a proper technique that significantly improves toxoplasmosis diagnosis. However, a more sensitive technique is required. This study compared real-time PCR with nested PCR using B1, SAG-4, and MAG-1 bradyzoite genes to diagnose toxoplasmosis in toxoplasmic retinochoroiditis patients. Methods Blood samples were collected from 10 patients with active toxoplasmic chorioretinal lesions and 10 healthy individuals. Blood samples including peripheral blood mononuclear cells (PBMCs), serum and whole blood samples were used for DNA extraction. Serum was also used to detect anti-toxoplasma IgG and IgM antibodies. Nested PCR and real-time PCR were performed using B1, SAG-4, and MAG-1 target genes. Results Five (50%) out of the 10 patients were tested positive for toxoplasmosis with nested PCR using the PBMC samples. All the five patients tested positive with nested PCR were also tested positive for toxoplasmosis with real-time PCR using the PBMC samples. The real-time PCR results demonstrated that 9(90%) out of the 10 patients were positive based on B1 and the remaining one (10%) was positive only based on MAG-1. In general, of the patients, five (50%) were positive using SAG-4 and three (30%) were positive in term of MAG-1 using PBMCs with real-time PCR. Conclusion It appears that PBMC samples have the best performance as the PCR extraction method and are a good source for toxoplasmosis diagnosis. The use of B22 and B23 target genes due to their high sensitivity and specificity along with bradyzoite genes are recommended for toxoplasmosis diagnosis using PBMC samples with real-time PCR.

Identification of semen and spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. In cases of alleged sexual assault where there is a long time gap between the incident and sample collection, or in cases of low sperm count, current methods have limitations of specificity, in the case of presumptive tests for semen, or the problem of recording spermatozoa by microscopy if they are few in number. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay using a spermatozoa-specific DNA methylated marker to identify spermatozoa has been reported previously by our laboratory. A key advantage over current methods is the increased sensitivity and specificity. A transition from a research tool to operational use requires blind trial testing and inter-laboratory trials. We report on a collaborative exercise where reagents of the 3-plex MSRE-PCR were sent to six participating laboratories. Each laboratory used their own equipment, consumables, and the presumptive reagents conventionally for body fluid (such as acid phosphatase or PSA), DNA extraction, and quantification in practical casework. The reagents and protocol for the 3-plex MSRE-PCR assay and 9 samples were provided by the organizing laboratory. The participating laboratories were requested to fill in the questionnaire after testing. The reported results from all the six participating laboratories were concordant and the expected correct results for the presence of spermatozoa. These outcomes verified the reproducibility and feasibility of the 3-plex MSRE-PCR assay. The results also indicated that the 3-plex MSRE-PCR assay was readily accessible to forensic laboratories for integrating it into current forensic casework processes.

Extraction of high quantity and quality DNAs from marine sponges, which contain diverse and abundant microbial communities, is important to molecular biology techniques for the analysis of nucleic acids. Several marine sponges and their associated microorganisms have been known to produce cytotoxic natural products on several cancer cell lines via DNA damage mechanisms. These marine cytotoxic substances might be one of the factors that cause the low quantity and quality of DNAs during the DNA extraction from its living origin. Therefore, the extraction of DNA of a Thai blue marine sponge Xestospongia sp. with sufficient purity and quantity for molecular study can be challenging. In this study, we developed an efficient extraction method to prepare DNAs from a Thai blue marine sponge Xestospongia sp. which accumulated a highly potent cytotoxic alkaloid with DNA-damaging activity, named Renieramycin M (RM), as a major constituent in high quantity. We demonstrated that removal of RM from the sponge samples by a simple methanolic extraction before DNA extraction dramatically increased the yield and purity of DNAs compared to the RM-unremoved sponge samples. High molecular weight (HMW) genomic DNA was obtained from sponge samples with 8 times of RM elimination by using modified NaOAc salting-out extraction method. The quantity and quality of the prepared DNAs were comparatively determined via spectrophotometry, electrophoresis, and 16S rRNA gene amplification. Our result suggests that the removal of DNA-damaging constituents from the samples is a crucial step and must be seriously taken as the necessary consideration for the practical protocol of DNA extraction.

Determining the diet of flying-foxes can increase understanding of how they function as pollinators and seed dispersers, as well as managing any negative impacts of large roosts. Traditional methods for diet analysis are time consuming, and not feasible to conduct for hundreds of animals. In this study, we optimised a method for diet analysis, based on DNA metabarcoding of environmental DNA (eDNA) from pollen and other plant parts in the faeces. We found that existing eDNA metabarcoding protocols are suitable, with the most useful results being obtained using a commercial food DNA extraction kit, and sequencing 350–450 base pairs of a DNA barcode from the internally transcribed spacer region (ITS2), with ~550 base pairs of the chloroplast rubisco large subunit (rbcL) as a secondary DNA barcode. A list of forage plants was generated for the little red flying-fox (Pteropus scapulatus), the black flying-fox (Pteropus alecto) and the spectacled flying-fox (Pteropus conspicillatus) from our collection sites across Queensland. The diets were determined to comprise predominantly Myrtaceae species, particularly those in the genera Eucalyptus, Melaleuca and Corymbia. With more plant genomes becoming publicly available in the future, there are likely to be further applications of eDNA methods in understanding the role of flying-foxes as pollinators and seed dispersers.

Undergraduate laboratory courses are essential to teaching core principles in STEM. This course, Quantitative Biological Methods, provides a unique approach to teaching molecular biology research techniques to students, in a laboratory that is delivered in a sequence that parallels standard biomedical research laboratory protocols. Students attend a lecture where they are taught the essential principles of biomedical research, and a lab where they learn to use laboratory equipment, perform experiments, and purify and quantify DNA and proteins. The course begins with an introduction to laboratory safety, pipetting, centrifugation, spectrophotometry, and other basic laboratory techniques. Next, the lab focuses on the purification and analysis of glutathione S-transferase (GST) fused to green fluorescent protein (GFP) from an Escherichia coli lysate. Students study this GST-GFP fusion protein and perform protein quantification, enzyme assays, chromatography, fluorescent detection, normalization, SDS-PAGE, and western blotting. Students then learn recombinant DNA technology using the GST-GFP vector that was the source of the fusion protein in the prior labs, and perform ligation, transformation of E. coli cells, blue/white screening, DNA purification via a miniprep, PCR, DNA quantification, restriction enzyme digestion, and agarose gel electrophoresis. Students write laboratory reports to demonstrate an understanding of the principles of the laboratory methods, and they must present and critically analyze their data. The lab methods described herein aim to emphasize the core molecular biology principles and techniques, prepare students for work in a biomedical research laboratory, and introduce students to both GST and GFP, two versatile laboratory proteins.