Short DNA Fragments Research Articles

Direct Capture and Amplification of Small Fragmented DNAs Using Nitrogen-Mustard-Coated Microbeads.

Circulating cell-free DNA (cfDNA) has been implicated as an important biomarker and has been intensively studied for "liquid biopsy" applications in cancer diagnostics. Owing to its small fragment size and its low concentration in circulation, cfDNA extraction and purification from serum samples are complicated, and the extraction yield affects the precision of subsequent molecular diagnostic tests. Here, we report a novel approach using nitrogen-mustard-coated DNA capture beads (NMD beads) that covalently capture DNA and allow direct subsequent polymerase chain reaction (PCR) amplification from the NMD bead without elusion. The complex DNA extraction and purification processes are not required. To illustrate the diagnostic use of the NMD beads, we detected short DNA fragments (142 bp) that were spiked into fetal bovine serum (as a model serum sample). The spiked DNAs were captured directly from serum samples and detected using real-time PCR at concentrations as low as 10 fg/mL. We anticipate that this DNA capture bead technique has the potential to simplify the preanalytical processes required for cfDNA detection, which could significantly expand the diagnostic applications of liquid biopsy.

Standardized Comparison of Different DNA Sequencing Platforms

Next-generation sequencing (NGS) has largely been implemented for clinical testing in many laboratories and offers a wide variety of applications ranging from targeted oncology panels to metagenomic sequencing for bacterial classification to population-based whole genome sequencing. Most laboratories operate NGS platforms that use short-read (SR) sequencing chemistry that sequences short fragments of DNA (reads) that are up to a few hundreds of base pairs in length. The reads are then reassembled to a reference genome assembly to detect genetic alterations such as single nucleotide variations (SNVs) or insertion/deletions (INDELs). Third-generation sequencing technology has emerged that sequences long reads (LR) that are several thousands of base pairs in length that can also detect larger structural alterations (e.g., translocations, inversions, duplications). An advantage of LR sequencing is the ability to resolve difficult-to-sequence regions of the genome where large stretches of repetitive sequences are present that can be challenging to align to the correct portion of the reference genome. A large comprehensive comparison of SR and LR technology, within the context of clinical laboratory testing, had not been undertaken until recently.

A sequence-based two-layer predictor for identifying enhancers and their strength through enhanced feature extraction.

Enhancers are short regulatory DNA fragments that are bound with proteins called activators. They are free-bound and distant elements, which play a vital role in controlling gene expression. It is challenging to identify enhancers and their strength due to their dynamic nature. Although some machine learning methods exist to accelerate identification process, their prediction accuracy and efficiency will need more improvement. In this regard, we propose a two-layer prediction model with enhanced feature extraction strategy which does feature combination from improved position-specific amino acid propensity (PSTKNC) method along with Enhanced Nucleic Acid Composition (ENAC) and Composition of k-spaced Nucleic Acid Pairs (CKSNAP). The feature sets from all three feature extraction approaches were concatenated and then sent through a simple artificial neural network (ANN) to accurately identify enhancers in the first layer and their strength in the second layer. Experiments are conducted on benchmark chromatin nine cell lines dataset. A 10-fold cross validation method is employed to evaluate model's performance. The results show that the proposed model gives an outstanding performance with 94.50%, 0.8903 of accuracy and Matthew's correlation coefficient (MCC) in predicting enhancers and fairly does well with independent test also when compared with all other existing methods.

DNA‐Based Nanoarchitectures as Eminent Vehicles for Smart Drug Delivery Systems

AbstractIn order to cure diseases efficiently with minimal side effects in sophisticated medicine, eminent drugs must be selectively delivered to diseased parts and allow to work only there. However, most of drug delivery systems (DDSs) previously proposed are not yet sufficiently selective and effective. This review covers recent developments of DDS in which DNA nanoarchitectures are employed as drug carriers. From short DNA fragments, a variety of complicated nanomedicines are fabricated to show unprecedentedly smart DDS functions. Encapsulated drugs are released mostly through controlled disassembly of DNA nanoarchitectures. Recent attention has been focusing to the use of appropriate stimuli to regulate each process in the DDS (delivery of drug, its release, and others) for further improved therapy. As intracellular stimuli for the regulation, various endogenous compounds (e.g., mRNA and enzymes), which are specifically abundant in target cells, are employed. Alternatively, physical perturbation (e.g., light irradiation) is provided from outside of tissues to control DDS. By combining multiple strategies in terms of new concepts, enormously smart DDSs are being achieved. Further progresses of these approaches in the future are guaranteed by complete freedom of molecular design in DNA nanoarchitectonics.

Aptamers-Diagnostic and Therapeutic Solution in SARS-CoV-2.

The SARS-CoV-2 virus is currently the most serious challenge to global public health. Its emergence has severely disrupted the functioning of health services and the economic and social situation worldwide. Therefore, new diagnostic and therapeutic tools are urgently needed to allow for the early detection of the SARS-CoV-2 virus and appropriate treatment, which is crucial for the effective control of the COVID-19 disease. The ideal solution seems to be the use of aptamers—short fragments of nucleic acids, DNA or RNA—that can bind selected proteins with high specificity and affinity. They can be used in methods that base the reading of the test result on fluorescence phenomena, chemiluminescence, and electrochemical changes. Exploiting the properties of aptamers will enable the introduction of rapid, sensitive, specific, and low-cost tests for the routine diagnosis of SARS-CoV-2. Aptamers are excellent candidates for the development of point-of-care diagnostic devices and are potential therapeutic tools for the treatment of COVID-19. They can effectively block coronavirus activity in multiple fields by binding viral proteins and acting as carriers of therapeutic substances. In this review, we present recent developments in the design of various types of aptasensors to detect and treat the SARS-CoV-2 infection.

Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments.

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

A modular and reconfigurable open-channel gated device for the electrokinetic extraction of cell-free DNA assays

The high-efficiency separation and extraction of short fragments of cell-free DNA (cfDNA) remain challenging due to their low abundance and short lengths. This study presents a method for separating short cfDNA fragments, with lengths ranging from about 100 to 200 base pairs, from liquid human plasma samples into separable and extractable bands as solid agarose gel slabs. To achieve this, a novel millimeter-scale fluidic device is used for sample handling, transient isotachophoresis, and extraction. The device features open-to-atmosphere liquid chambers that define and manually actuated (i.e., movable) agarose-made gate valve structures. The agarose gates then define discrete zones for buffers, sample injection, DNA pre-concentration via isotachophoresis, size-based gel separation, and DNA-band extraction. As a demonstration of its efficacy, the device is applied to the enrichment and purification of M. tuberculosis genomic DNA fragments spiked in human plasma samples. This purified cfDNA is analyzed using the quantitative polymerase chain reaction (qPCR) of the IS6110 repetitive sequence in the M. tuberculosis genome. The data from this study demonstrates that high sensitivity can be achieved in cfDNA detection, as shown by the comparison with a typical solid-phase extraction method and buffer spiked with cfDNA. Evidence is presented that suggests plasma peptides generated by treatment of the sample with proteinase K acts as endogenous spacer molecules, which improve the resolution and purification of DNA relative to the marker dye and other contaminants that decrease the signal level in qPCR.

EDNA profiling of mammals, birds, and fish of surface waters by mitochondrial metagenomics: application for source tracking of fecal contamination in surface waters.

Knowing the composition of animals present in aquatic ecosystems can tell us about the anthropic pressures on these environments. One of these pressures is the occurrence of fecal contamination. However, this contamination can originate from more than one animal species in areas where urban and agricultural activities overlap. Mitochondrial DNA (mtDNA) has become the standard barcoding tool to identify the presence of animal species in environment. Amplicon-sequencing metagenomics is a powerful approach to derive the animal profile in an environment. However, PCR primers targeting mtDNA of a broad range of animals are highly degenerate or generate short DNA fragments that could cause ambiguous affiliation. Here we report the development of a new set of primers targeting the mitochondrial 16S ribosomal RNA genes of a broad range of terrestrial and aquatic animals, which include mammals, birds, and fishes. These primers successfully amplified mtDNA from environmental DNA (eDNA) extracted from surface waters. Sequencing the resulting amplicons revealed the presence of mammals and birds that may contribute in fecal contamination of surface water. In one of the river samples high in fecal indicator bacteria, human and bovine mtDNA accounted for 40.5% and 4.1% of the sequences, respectively, suggesting fecal contamination by these two animals. These findings indicate that our PCR primers coupled with amplicon-sequencing metagenomics contribute in profiling the animal diversity in the surface waters and its surrounding. This approach could be a valuable tool to identify simultaneously the potential contribution of various animals as sources of fecal contamination in surface waters.

Circulating Cell Free DNA Integrity Index as a Biomarker for Response to Chemotherapy in Patients with Metastatic Colorectal Carcinoma.

Objective:Assessing plasma Cell Free DNA (cfDNA) integrity index as a biomarker for response prediction and early response evaluation in mCRC patients receiving chemotherapy, in comparison to Carcinoembryonic antigen (CEA) and Carbohydrate antigen 19-9 (CA19-9), to be used as an additional tool to computed tomography (CT). Methods:CEA, CA19-9, cfDNA concentration and cfDNA integrity index (ALU 247/115) measurements were conducted on 86 subjects divided into 43 healthy volunteers and 43 mCRC patients, before starting chemotherapy and then after 6-12 weeks of therapy initiation (3-4 cycles FOLFOX) at first response assessment. Plasma cfDNA integrity index was calculated as the ratio of long to short DNA fragments (ALU 247/115) amplified and detected by real-time PCR. Serum CEA and CA19-9 were measured by chemiluminescent immunometric assay. Results:Baseline cfDNA integrity index was statistically significantly different between responders and non-responders (p=0.03). It was found that at cut off 0.608, sensitivity was 73.7%, specificity was 66.7% and diagnostic accuracy=69.77%. Markers with statistical significant difference between responders and non-responders after chemotherapy were CEA % change (p=0.035), CA19-9 (p=0.024), cfDNA integrity index (p=0.035) and cfDNA integrity index % change (p<0.001). Among these markers, cfDNA integrity index % change had the best sensitivity (84.2%), specificity (95.2%) and diagnostic accuracy (90.7%) at cut off -17.827%. Conclusion: Baseline cfDNA integrity index can be used as a potential marker to predict response to chemotherapy. cfDNA integrity index (ALU 247/115) % change rather than its absolute value is superior to CEA, CA19-9, cfDNA concentration and their % changes in early assessment of response to chemotherapy.

A systematic genome-wide account of binding sites for the model transcription factor Gcn4

Sequence-specific DNA-binding transcription factors are central to gene regulation. They are often associated with consensus binding sites that predict far more genomic sites than are bound in vivo. One explanation is that most sites are blocked by nucleosomes, such that only sites in nucleosome-depleted regulatory regions are bound. We compared the binding of the yeast transcription factor Gcn4 in vivo using published ChIP-seq data (546 sites) and in vitro, using a modified SELEX method (“G-SELEX”), which utilizes short genomic DNA fragments to quantify binding at all sites. We confirm that Gcn4 binds strongly to an AP-1-like sequence (TGACTCA) and weakly to half-sites. However, Gcn4 binds only some of the 1078 exact matches to this sequence, even in vitro. We show that there are only 166 copies of the high-affinity RTGACTCAY site (exact match) in the yeast genome, all occupied in vivo, largely independently of whether they are located in nucleosome-depleted or nucleosomal regions. Generally, RTGACTCAR/YTGACTCAY sites are bound much more weakly and YTGACTCAR sites are unbound, with biological implications for determining induction levels. We conclude that, to a first approximation, Gcn4 binding can be predicted using the high-affinity site, without reference to chromatin structure. We propose that transcription factor binding sites should be defined more precisely using quantitative data, allowing more accurate genome-wide prediction of binding sites and greater insight into gene regulation.

Historical DNA from Museum Shell Collections: Evaluating the Suitability of Dried Micromollusks for Molecular Systematics

Natural history collections are an invaluable resource that can inform systematic studies and biodiversity discovery, and also contribute to understanding changes in species abundance and distributions over time. The decline in abundance and diversity of Pacific Island land snails has been a major conservation concern for more than six decades, but only the largest and most colorful snails are protected under the US Endangered Species act, and few are listed as critically endangered on the IUCN Red List. Like most invertebrates, the conservation status of many Hawaiian land snails still need assessment. Molecular data are highly informative for revising species limits and understanding evolutionary patterns and processes, but with as much as 70% of Hawaiian land snails already extinct, few fresh samples are available from which to extract DNA. To overcome the lack of material suitable for DNA barcoding, we test whether short DNA fragments of 225 to 355 bp can be sequenced from museum snail shells containing dried tissues collected more than 50 years ago. Short DNA sequences (225 bp) were obtained from 66.7% of lots, while longer DNA sequences (355 bp) were successfully sequenced from 24.2% of lots. Snail specimens stored in natural history cabinets for more than 100 years were successfully sequenced, supporting the inclusion of these materials for modern biodiversity studies. Molecular data from this study represents a small proportion of Hawaiian microsnail species housed among the millions of specimens in the Bishop Museum in Hawaii and other natural history collections. Additional resources and focused efforts are needed to scale this approach to incorporate many more of the hundreds of snail species in need of assessment in Hawaii. More broadly, there are large representative collections of endangered Pacific Island non-marine snails in many natural history museums that may be suitable for molecular work, either with DNA barcoding or other genomic approaches.

Noninvasive Prenatal Test for β-Thalassemia and Sickle Cell Disease Using Probe Capture Enrichment and Next-Generation Sequencing of DNA in Maternal Plasma.

Noninvasive prenatal testing (NIPT) of chromosomal aneuploidies based on next-generation sequencing (NGS) analysis of fetal DNA in maternal plasma is well established, but testing for autosomal recessive disorders remains challenging. NGS libraries prepared by probe capture facilitate the analysis of the short DNA fragments plasma. This system has been applied to the β-hemoglobinopathies to reduce the risk to the fetus. Our probe panel captures >4 kb of the HBB region and 435 single-nucleotide polymorphisms (SNPs) used to estimate fetal fraction. Contrived mixtures of DNA samples, plasma, and whole blood samples from 7 pregnant women with β-thalassemia or sickle cell anemia mutations and samples from the father, sibling, and baby or chorionic villus were analyzed. The fetal genotypes, including point mutations and deletions, were inferred by comparing the observed and expected plasma sequence read ratios, based on fetal fraction, at the mutation site and linked SNPs. Accuracy was increased by removing PCR duplicates and by in silico size selection of plasma sequence reads. A probability was assigned to each of the potential fetal genotypes using a statistical model for the experimental variation, and thresholds were established for assigning clinical status. Using in silico size selection of plasma sequence files, the predicted clinical fetal genotype assignments were correct in 9 of 10 plasma libraries with maternal point mutations, with 1 inconclusive result. For 2 additional plasmas with deletions, the most probable fetal genotype was correct. The β-globin haplotype determined from linked SNPs, when available, was used to infer the fetal genotype at the mutation site. This probe capture NGS assay demonstrates the potential of NIPT for β-hemoglobinopathies.

Platelet Factor 4 (PF4) Modulates the Prothrombotic Nature of Neutrophil-Extracellular Traps (NETs): Therapeutic Implications of a NET-Stabilization Strategy

Sepsis is a dysregulated response to infection leading to life-threating organ damage. Although it remains one of the most common causes of mortality worldwide, it lacks targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens and prevent bacterial dissemination. However, when NETs are degraded by circulating nucleases they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger coagulation, induce complement activation, and cause oxidative tissue damage. We proposed a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4. Binding of PF4 enhances NET DNase-resistance, promotes NDP sequestration and increases bacterial capture, improving survival in murine sepsis.As NETs are considered prothrombotic, we were concerned that NET stabilization may increase the risk of clot formation. We therefore sought to determine the effect of PF4-NET stabilization on the thrombogenicity of NETs to learn if this strategy is safe for clinical application. To that end, we examined the effect of PF4 on the thrombotic potential of DNA and NET fragments at different states of nuclease digestion. High molecular weight (hmw) genomic DNA (hmwDNA, >50kbp) was isolated from human whole blood. hmwDNA was digested with restriction enzymes (EcoRI and AluI) for 15min to generate DNA fragments of ~4kbp and ~250bp, respectively. Neutrophils were also isolated from human blood and stimulated with 100 nM PMA to produce neutrophil-adherent NETs, which were cleaved from cell bodies by treatment with 4U/mL DNase I for 20 minutes, releasing NETs >50kbp (hmwNETs). Additional incubation of hmwNETs with DNase I yielded smaller NET fragments. We assessed in vitro activation of coagulation by DNA and NETs by measuring thrombin generation and fibrin formation in platelet-poor plasma using fluorogenic substrate and turbidity assays.Neutrophil-adherent NETs induced far less thrombin generation and fibrin formation in plasma than hmwDNA and hmwNETs. PF4 significantly increased lag time and reduced peak thrombin formation induced by both hmwDNA and hmwNETs. Binding of PF4 also delayed clot initiation time and reduced the rate of fibrin generation. Digestion of hmwDNA and hmwNETs to smaller fragments markedly enhanced thrombogenicity.We posited that shorter DNA fragments are more thrombogenic because they have a greater proportion of end-fragment DNA that exposes more single-stranded DNA. To test this hypothesis, we subjected hmwDNA and digested DNA to heat denaturation at 95°C and rapid cooling to generate single stranded DNA and found that this accelerated fibrin generation. Although the anti-thrombotic effect of PF4 was most pronounced with longer DNA and NET fragments, it continued to significantly reduce fibrin generation induced by shorter DNA fragments, perhaps by stabilizing the fragments to prevent exposure of single-stranded DNA.In conclusion, although prior studies have shown that NETs increase the risk of thrombosis in sepsis, we propose the counter-intuitive concept that PF4-stabilization decreases the risk of NET-mediated prothrombotic state by (1) inhibiting DNase cleavage of intact NETs and subsequent liberation of prothrombotic cfDNA from non-thrombogenic neutrophil-adherent NETs, and (2) preventing further digestion of circulating cfDNA into shorter and more prothrombotic fragments. Although NETs are a double-edged sword: capable of capturing pathogens but inducing host-tissue damage and thrombosis when degraded, treatment with PF4 tips the balance, limiting the capacity of NETs to induce fibrin generation and thrombosis, while enhancing their ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis and merits further investigation in translational studies. DisclosuresNo relevant conflicts of interest to declare.

Proximal-end bias from in-vitro reconstituted nucleosomes and the result on downstream data analysis.

The most basic level of eukaryotic gene regulation is the presence or absence of nucleosomes on DNA regulatory elements. In an effort to elucidate in vivo nucleosome patterns, in vitro studies are frequently used. In vitro, short DNA fragments are more favorable for nucleosome formation, increasing the likelihood of nucleosome occupancy. This may in part result from the fact that nucleosomes prefer to form on the terminal ends of linear DNA. This phenomenon has the potential to bias in vitro reconstituted nucleosomes and skew results. If the ends of DNA fragments are known, the reads falling close to the ends are typically discarded. In this study we confirm the phenomenon of end bias of in vitro nucleosomes. We describe a method in which nearly identical libraries, with different known ends, are used to recover nucleosomes which form towards the terminal ends of fragmented DNA. Finally, we illustrate that although nucleosomes prefer to form on DNA ends, it does not appear to skew results or the interpretation thereof.

Short single-stranded DNAs with putative non-canonical structures comprise a new class of plasma cell-free DNA

BackgroundCell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations.ResultsWe aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments.ConclusionsWe propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

Exploring DNA preservation and taxonomic diversity in postmedieval human tooth samples in Latvia

Ancient DNA (aDNA) provides unique opportunities to explore various evolutionary and ecological processes. In particular, aDNA studies offer new possibilities for the in-depth investigation of historical social structures at local and regional scales, reconstruction of ancient human microbiomes, and identification of ancient pathogens. The success of these studies depends on the preservation of aDNA, as well as on our ability to distinguish between historical DNA fragments, either human, animal, plant or microbial, and contaminant modern environmental DNA. The aim of this study was to examine the preservation of aDNA in human postmedieval archaeological tooth samples in Latvia dated 15th–17th century CE. The taxonomic composition of the short DNA fragment fractions was compared to that of the total DNA samples to explore the presence and diversity of environmental bacteria in aDNA datasets.Samples were analyzed by a shotgun metagenomics-based approach. The taxonomic profiles of metagenomics data revealed that the majority of microbial phyla/genera/species belonged to the typical soil microbiota, indicating the contamination of archaeological samples by environmental microorganisms. No significant differences in alpha or beta diversity indices were found between the short and total DNA fractions. The presence of soil microbiota in the short DNA fractions and the discordance between the microbial patterns suggested the fragmentation of the historical environmental DNA. The proportions of both human and oral microbial DNA were significantly higher in the short DNA fragment libraries than in the total DNA samples, and DNA reads showed characteristic damage patterns, suggesting severe degradation of endogenous aDNA. The human aDNA yield obtained in this study was sufficient to perform molecular sex typing, and the genetic results confirmed morphological data in all cases.Overall, sufficient preservation of endogenous aDNA was detected in human archaeological tooth samples in Latvia dated 15th–17th century CE, which enables future studies on early-modern populations and pre-industrial oral microbial communities. The analysis of total DNA samples along with short DNA fragments may provide additional information, such as the microbial composition of the burial environment. Concomitant analysis of historical and modern environmental DNA could provide additional data for deciphering the complete ancient microbiome profiles.

Universal performance of benzalkonium chloride for the preservation of environmental DNA in seawater samples

AbstractEnvironmental DNA (eDNA) analysis allows noninvasive and cost‐effective monitoring of macroorganisms' distribution and composition in aquatic ecosystems. Benzalkonium chloride (BAC) is an inexpensive and simple preservative for eDNA in water samples and has been used in many eDNA studies for preventing its degradation during transportation. However, its preservation performance has limitedly been evaluated by species‐specific assays, targeting short fragments of mitochondrial DNA in freshwater and brackish ecosystems. Here, we examined the performance of BAC in preserving eDNA in seawater samples, targeting different fragment lengths of mitochondrial and nuclear eDNA, and community information inferred by eDNA metabarcoding. We quantified the time series changes of Japanese jack mackerel (Trachurus japonicus) eDNA concentrations in experimental tanks and inshore seawater to compare the yields and decay rates of eDNA between BAC treatments. For both tank and field samples, BAC treatment substantially suppressed the degradation of all types of target eDNA and increased the eDNA yields at the start of the experiment. In addition, we performed eDNA metabarcoding targeting fish community to compare the species richness and composition in seawater samples between BAC treatments, showing that the number of fish species in field samples hardly varied throughout a day by BAC treatment. Our findings indicate high versatility of BAC in preserving both the quantitative (copy number) and the qualitative (species richness) information on various types of aqueous eDNA in various environmental conditions. BAC should therefore be used to minimize the false‐negative detection of eDNA, regardless of target genetic regions, fragment sizes, environmental conditions, and detection strategies.

Linking the state of environmental DNA to its application for biomonitoring and stock assessment: Targeting mitochondrial/nuclear genes, and different DNA fragment lengths and particle sizes

AbstractEnvironmental DNA (eDNA) analysis is a revolutionary tool for non‐invasive, cost‐effective, and highly sensitive monitoring of species distribution and abundance; however, there remain some uncertainties related to eDNA detection and quantification, as well as limitations in terms of its ecological interpretation. Although these may be elucidated by better understanding the characteristics and dynamics of eDNA, insight into such basic eDNA information has been limited in this decade, contrary to the advancements in eDNA applications targeting various taxa and environments. This review compiled previous findings regarding the characteristics and dynamics of macrobial eDNA and provides insights into how the basic knowledge of eDNA can contribute to the refinement and development of eDNA analysis for biomonitoring and stock assessment. A literature survey revealed that studies on the cellular and molecular state of eDNA were particularly lacking (18/728 papers from 2008 to 2020), resulting in a limited understanding regarding the process of eDNA transport and degradation. This review highlighted a number of studies targeting various types of eDNA beyond short mitochondrial DNA fragments (nuclear eDNA, longer eDNA fragments, and larger eDNA particles) to show how information on the state of eDNA improves the reliability of species detection and accuracy of abundance estimation, as well as provide more detailed information on individuals other than their presence and abundance. Linking the state of eDNA to its application will advance the analysis of eDNA and improve its application as a tool for monitoring biodiversity, ecosystem function, and fisheries resources.

Fast Fabrication of Solid-State Nanopores for DNA Molecule Analysis.

Solid-state nanopores have been developed as a prominent tool for single molecule analysis in versatile applications. Although controlled dielectric breakdown (CDB) is the most accessible method for a single nanopore fabrication, it is still necessary to improve the fabrication efficiency and avoid the generation of multiple nanopores. In this work, we treated the SiNx membranes in the air–plasma before the CDB process, which shortened the time-to-pore-formation by orders of magnitude. λ-DNA translocation experiments validated the functionality of the pore and substantiated the presence of only a single pore on the membrane. Our fabricated pore could also be successfully used to detect short single-stranded DNA (ssDNA) fragments. Using to ionic current signals, ssDNA fragments with different lengths could be clearly distinguished. These results will provide a valuable reference for the nanopore fabrication and DNA analysis.

Diffusion of Short Semiflexible DNA Polymer Chains in Strong and Moderate Confinement.

In many technological applications, DNA is confined within nanoenvironments that are smaller than the size of the unconfined polymer in solution. However, the dependence of the diffusion coefficient on molecular weight and characteristic confinement dimension remains poorly understood in this regime. Here, convex lens-induced confinement (CLiC) was leveraged to examine how the diffusion of short DNA fragments varied as a function of slit height by using single-molecule fluorescence tracking microscopy. The diffusion coefficient followed approximate power law behavior versus confinement height, with exponents of 0.27 ± 0.01, 0.32 ± 0.02, and 0.42 ± 0.06 for 692, 1343, and 2686 base pair chains, respectively. The weak dependence on slit height suggests that shorter semiflexible chains may adopt increasingly rodlike conformations and therefore experience weaker excluded-volume interactions as the confinement dimension is reduced. The diffusion coefficient versus molecular weight also exhibited apparent power law behavior, with exponents that varied slightly (from -0.89 to -0.85) with slit height, consistent with hydrodynamic interactions intermediate between Rouse and Zimm model predictions.