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Sedimentary ancient DNA (sedaDNA) has been established as a viable biomolecular proxy for tracking taxon presence through time in a local environment, even in the total absence of surviving tissues. SedaDNA is thought to survive through mineral binding, facilitating long-term biomolecular preservation, but also challenging DNA isolation. Two common limitations in sedaDNA extraction are the carryover of other substances that inhibit enzymatic reactions, and the loss of authentic sedaDNA when attempting to reduce inhibitor co-elution. Here, we present a sedaDNA extraction procedure paired with targeted enrichment intended to maximize DNA recovery. Our procedure exhibits a 7.7–19.3x increase in on-target plant and animal sedaDNA compared to a commercial soil extraction kit, and a 1.2–59.9x increase compared to a metabarcoding approach. To illustrate the effectiveness of our cold spin extraction and PalaeoChip capture enrichment approach, we present results for the diachronic presence of plants and animals from Yukon permafrost samples dating to the Pleistocene-Holocene transition, and discuss new potential evidence for the late survival (~9700 years ago) of mammoth (Mammuthus sp.) and horse (Equus sp.) in the Klondike region of Yukon, Canada. This enrichment approach translates to a more taxonomically diverse dataset and improved on-target sequencing.

Background : Automation has increasingly become more commonplace in the research laboratory workspace. The introduction of articulated robotic arms allows the researcher more flexibility in the tasks a single piece of automated machinery can perform. We set out to incorporate automation in processing of genomic DNA organic extractions to increase throughput and limit researchers to the exposure of organic solvents. Methods : In order to automate the genome sequencing pipeline in our laboratory, we programmed a dual-arm anthropomorphic robot, the Robotic Biology Institute's Maholo LabDroid, to perform organic solvent-based genomic DNA extraction from cell lysates. To the best of our knowledge, this is the first time that automation of phenol-chloroform extraction has been reported. Results: We achieved routine extraction of high molecular weight genomic DNA (>100 kb) from diverse biological samples including algae cultured in sea water, bacteria, whole insects, and human cell lines. The results of pulse-field electrophoresis size analysis and the N50 sequencing metrics of reads obtained from Nanopore MinION runs verified the presence of intact DNA suitable for direct sequencing. Conclusions : We present the workflow that can be used to program similar robots and discuss the problems and solutions we encountered in developing the workflow. The protocol can be adapted to analogous methods such as RNA extraction, and there is ongoing work to incorporate further post-extraction steps such as library construction. This work shows the potential for automated robotic workflows to free molecular biological researchers from manual interventions in routine experimental work. A time-lapse movie of the entire automated run is included in this report.

Metabarcoding of invertebrate‐derived DNA (iDNA) is an excellent tool for assessing terrestrial mammal diversity. A major constraint to wider adoption is the time and costs associated with sample processing. Here, we explored the effectiveness of bulk fly iDNA extraction, which increased the pooling of flies by an order of magnitude over currently used methods. One extraction method involved removing a single leg per fly from many flies, pooling these legs, and destructively extracting DNA from the pool (Nflies legs per pool = 105). The other involved non‐destructively extracting DNA from a large pool of entire flies (Nflies per pool = 105). We tested these methods on flies collected at eight sites, representing three different habitats across five countries in sub‐Saharan Africa (Nflies/site = 105; Nflies total = 840). We compared the mammal species detected using metabarcoding of these extracts with extracts generated using an approach that is currently widely used, the cost‐ and labor‐intensive destructive extraction of small pools of entire flies (Nflies per pool = 7; Nflies per site = 105 flies). The non‐destructive extraction from large pools of entire flies detected a greater number of mammals (Nspecies detections total = 58; x¯species/site $$ {\\overline{x}}_{\\mathrm{species}/\\mathrm{site}} $$  = 7.3; range = 1–12) than the destructive extraction from large pools of fly legs (Nspecies detections total = 15; x¯species/site $$ {\\overline{x}}_{\\mathrm{species}/\\mathrm{site}} $$  = 1.9; range = 0–3), while detecting a similar number of mammals as the destructive extraction from small pools of entire flies (Nspecies detections total = 67; x¯species/site $$ {\\overline{x}}_{\\mathrm{species}/\\mathrm{site}} $$  = 8.4; range = 5–15). Our findings indicate that the non‐destructive extraction of large pools of entire fly bodies has the potential to streamline and reduce costs of fly iDNA extraction. We hope this will promote the use of fly iDNA in terrestrial biomonitoring efforts. Metabarcoding of invertebrate‐derived DNA (iDNA) is an excellent tool for assessing terrestrial mammal diversity, but the time and costs associated with sample processing constrain its wider adoption. Our study indicates that the non‐destructive extraction of large pools of entire fly bodies has the potential to streamline and reduce the costs of fly iDNA extraction.

Techniques for the extraction and use of nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissues, preserved over long time periods in libraries, have been developed. However, DNA extracted from FFPE tissues is generally damaged, and long-term storage may affect DNA quality. Therefore, it is important to elucidate the effect of long-term storage on FFPE tissues and evaluate the techniques used to extract DNA from them. In the present study, the yield, purity, and integrity of DNA in FFPE tissue samples was evaluated. Two DNA extraction techniques were used: A silica-binding DNA collection method using QIAamp DNA FFPE Tissue kit (QIA) and a total tissue DNA collection method using a WaxFree DNA extraction kit (WAX). A total of 25 FFPE tissues from lung adenocarcinomas were studied, which had been surgically resected and fixed at Okayama University Hospital prior to examination and subsequent storage at room temperature for 0.5, 3, 6, 9 and 12 years. Extracted DNA was quantified using ultraviolet absorbance, fluorescent dye, and quantitative polymerase chain reaction (qPCR). The quality of the DNA was defined by the absorbance ratio of 260 to 280 nm (A260/280) and Q-score, which is the quantitative value of qPCR product size ratio. The results demonstrated that the yield of total DNA extracted using WAX was significantly greater than when QIA was used (P<0.01); however, DNA extracted using WAX included more contaminants and was significantly more fragmented compared with DNA extracted using QIA (P<0.01). Aging had no significant effect on absolute DNA yield or DNA purity, although it did significantly contribute to increased DNA degradation for both QIA and WAX extraction (QIA P=0.02, WAX P=0.03; 0.5 years vs. 3 years, QIA P<0.01, WAX P=0.03; 9 years vs. 12 years). Both extraction methods are viable depending on whether high yield or high quality of extracted DNA is required. However, due to the increased degradation with age, storage time limits the available DNA in FFPE tissues regardless of the extraction method.

The long‐read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource‐limited settings, such as field conditions or low‐ and middle‐income countries. For this purpose, protocols for extracting high‐molecular‐weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram‐negative bacteria. However, we found that without modification, these protocols are unsuitable for gram‐positive Streptococcus spp., a major threat to fish farming and food security in low‐ and middle‐income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high‐molecular‐weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish‐pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high‐molecular‐weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low‐copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource‐limited settings‐adapted protocol for high‐molecular‐weight DNA extraction could be implemented successfully for similarly recalcitrant‐to‐lysis gram‐positive bacteria, and it represents a method of choice for MinION‐based disease diagnostics in low‐ and middle‐income countries. A method for extracting high‐molecular‐weight DNA from Streptococcus spp., suitable for resource‐limited settings, was optimized. The extracted DNA was used for nanopore whole‐genome sequencing. This sequencing technique is valuable for the rapid diagnosis of diseases in warm water aquaculture, such as streptococcosis, under field conditions in low‐ and middle‐income countries. It provides near real‐time information on species, serotype, and the presence of plasmids. This was demonstrated by the discovery of a 15 kb low copy number mobilizable plasmid in Streptococcus iniae, the first reported in this species, which we named pSI1.

To reduce the morbidity and mortality of candidemia patients through rapid treatment, the development of a simple, rapid molecular diagnostic method that is based on nucleic acid extraction and is superior to conventional methods for detecting Candida in the blood is necessary. We developed a multiplex Candida Pan/internal control (IC) loop-mediated isothermal amplification (LAMP) assay and a simple DNA extraction boiling protocol using Chelex-100 that could extract yeast DNA in blood within 20 min. The Chelex-100/boiling method for DNA extraction showed comparable efficiency to that of the commercial QIAamp UCP Pathogen Mini Kit using Candida albicans qPCR. In addition, the Candida Pan/IC LAMP assay showed superior sensitivity to that of general Candida Pan and species qPCRs against clinical DNA samples extracted with the QIAamp UCP Pathogen Mini Kit and Chelex-100/boiling method. The Candida Pan/IC LAMP assay followed by Chelex-100/boiling-mediated DNA extraction showed high sensitivity (100%) and specificity (100%) against clinical samples infected with Candida. These results suggest that the Candida Pan/IC LAMP assay could be used as a rapid molecular diagnostic test for candidemia.

We present a rapid high-throughput DNA extraction method for use with EDTA-anticoagulated blood using silicon dioxide (SiO2) powder in a guanidine–HCl solution, hereinafter referred to as “Glassmilk.” The method utilizes a 96-well deep-well plate, enabling DNA extraction from 96 samples in under 3 h. The method integrates cell lysis, washing, elution, and DNA storage within the same well, eliminating the need for DNA transfer. The Glassmilk extraction method is cost-effective and fast, and it avoids expensive or toxic reagents by using only basic lab equipment. The method yielded approximately 40 μg of high-quality DNA from 200 μl of blood. The DNA yield of the Glassmilk method was about 50% higher, and the purity of the DNA was comparable to those obtained using two commercial column-based extraction kits that were used for comparison. The cost per sample was around $1, with the most expensive item being the filter pipette tips, which account for about $0.80 per sample. As we show, the extracted DNA is suitable for downstream applications such as polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism analysis, and qPCR. The method can be adapted for various sample types, including biopsies, fecal samples, cultured cells, and bacteria (see “subprotocols” section), and can also be applied in individual Eppendorf tubes. Our protocol may be useful for basic molecular research in laboratories having limited funds.

Genome sequencing is important for discovering critical genes in crops and improving crop breeding efficiency. Generally, fresh, young leaves are used for DNA extraction from plants. However, seeds, the storage form, are more efficient because they do not require cultivation and can be ground at room temperature. Yet, only a few DNA extraction kits or methods suitable for seeds have been developed to date. In this study, we introduced an improved (IMP) Boom method that is relatively low-cost, simple to operate, and yields high-quality DNA that can withstand long-read sequencing. The method successfully extracted approximately 8 µg of DNA per gram of seed weight from soybean seeds at an average concentration of 48.3 ng/µL, approximately 40-fold higher than that extracted from seeds using a common extraction method kit. The A260/280 and A260/230 values of the DNA were 1.90 and 2.43, respectively, which exceeded the respective quality thresholds of 1.8 and 2.0. The DNA also had a DNA integrity number value (indicating the degree of DNA degradation) of 8.1, higher than that obtained using the kit and cetyltrimethylammonium bromide methods. Furthermore, the DNA showed a read length N50 of 20.96 kbp and a maximum read length of 127.8 kbp upon long-read sequencing using the Oxford Nanopore sequencer, with both values being higher than those obtained using the other methods. DNA extracted from seeds using the IMP Boom method showed an increase in the percentage of the nuclear genome with a decrease in the relative ratio of chloroplast DNA. These results suggested that the proposed IMP Boom method can extract high-quality and high-concentration DNA that can be used for long-read sequencing, which cannot be achieved from plant seeds using other conventional DNA extraction methods. The IMP Boom method could also be adapted to crop seeds other than soybeans, such as pea, okra, maize, and sunflower. This improved method is expected to improve the efficiency of various crop-breeding operations, including seed variety determination, testing of genetically modified seeds, and marker-assisted selection.

Nucleic acid extraction is crucial for PCR detection of pathogenic bacteria to ensure food safety. In this study, a new magnetic extraction method was developed using 3D printing and magnetic silica beads (MSBs) to extract the target DNA from a large volume of bacterial sample and combined with microfluidic PCR to determine the bacteria. After proteinase K was added into a bacterial sample to lyse the bacteria and release the DNA, it was continuous-flow injected into the serpentine channel of the extraction chip, where magnetic silica bead chains had been formed in advance using a homogeneous magnetic field generated by two concentric semicircle magnets to capture the MSBs. Then, the flowing DNA was captured by the MSB chains, washed with alcohol, dried with gas, and eluted with deionized water to obtain the purified and concentrated DNA. Finally, the extracted DNA templates were injected into a microfluidic PCR chip with lyophilized amplification reagents and determined using a commercial qPCR device. The experimental results showed that the DNA extraction efficiency was more than 90%, and the lower detection limit of Salmonella was 102 CFU/mL. This new Salmonella detection method is promising to provide the rapid, sensitive, and simultaneous detection of multiple foodborne pathogens.

DNA extraction is usually the first step to perform molecular studies. This process can be nonviable due to genomic DNA (gDNA) extraction commercial kits prices. Furthermore, available DNA extraction protocols generally have high specificity, limiting their use to specific sources of biological material. In order to reduce costs, optimize time and laboratory logistics, besides to demonstrate a versatile protocol, the present study worked on an efficient DNA extraction protocol from somatic and non-somatic cells, using biological material from sheep as a model. For that, gDNA was extracted from whole blood, spermatozoa, and hair bulb cells, collected from three adult sheep, transported at 5ºC and stored at -20ºC until lab procedures. After extraction, gDNA concentration and purity were evaluated in a nano spectrophotometer. gDNA concentration from whole blood was greater (p < 0.05) than extracted from hair bulb cells, which in turn was superior (p < 0.05) than in spermatozoa. Also, gDNA from whole blood and, followed by, sperm showed greater (p < 0.05) purity when compared to gDNA of hair bulb cells. Adapting a gDNA extraction protocol, originally developed for bovine whole blood, enabled to obtain and isolate gDNA in different nucleated sheep cells