Low DNA Yield Research Articles

Extraction of Genomic DNA from Soil Samples by Polyethylene Glycol-Modified Magnetic Particles via Isopropanol Promotion and Ca2+ Mediation.

Obtaining reliable and informative DNA data from soil samples is challenging due to the presence of interfering substances and typically low DNA yields. In this work, we prepared poly(ethylene glycol)-modified magnetic particles (PEG@Fe3O4) for DNA purification. The particles leverage the facilitative effect of calcium ions (Ca2+), which act as bridges between DNA and PEG@Fe3O4 by coordinating with the phosphate groups of DNA and the hydroxyl groups on the particles. The addition of 2-propanol further enhances this Ca2+-mediated DNA adsorption by inducing a conformational change from the B-form to the more compact A-form of DNA. PEG@Fe3O4 demonstrates a DNA adsorption capacity of 144.6 mg g-1. When applied to the extraction of genomic DNA from soil samples, PEG@Fe3O4 outperforms commercial kits and traditional phenol-chloroform extraction methods in terms of DNA yield and purity. Furthermore, we developed a 16-channel automated DNA extraction device to streamline the process and reduce the extraction time. The successful amplification of target bacterial and fungal amplicons underscores the potential of this automated, device-assisted method for studying soil microbial diversity.

Liquid Biopsies in Follicular Thyroid Carcinomas-A Brief Report.

Thyroid cancer (TC) represents a significant health burden globally, with follicular thyroid cancer (FTC) posing diagnostic challenges despite advancements. This pilot study aimed to evaluate the utility of a liquid biopsy with cell-free DNA (cfDNA) in patients with FTC. Blood samples were collected from 13 patients diagnosed with FTC, DNA extraction was performed, and cfDNA was analyzed using the Illumina's TruSight Oncology 500 High-Throughput panel. The results revealed low tumor mutational burden and minimal pathogenic variants in cfDNA, indicating challenges such as low DNA yield and poor material quality despite adequate coverage. Our findings indicate that cfDNA as an add-on diagnostic tool in patients with FTC might not be a useful supplement.

Optimization of Total DNA Extraction from Dried Blood Samples.

While dried blood spots are a convenient source of genetic material, they are usually associated with a lower DNA yield than from a native sample. The study evaluated the DNA yield from dried blood samples prepared on glass fibre and cellulose membranes and investigated the reasons for the yield reduction. The extraction of total DNA from membrane-dried blood samples was optimized by spin-column extraction method. It was shown that preliminary short-term (20min) solubilization of a dried matrix in an aqueous medium, followed by standard extraction protocols for the mixture of the eluate with membranes, provides the highest DNA yield. The yield of DNA from a glass fibre membrane was 40-50% lower compared to a native sample, but on average, two times higher than from a conventional cellulose membrane (filter paper). The reduction of DNA yield when using a dried sample was found to be due to partial retention of nucleic acids by the membrane material during the lysis stage.

Comparison of microbial molecular diagnosis efficiency within unstable template metagenomic DNA samples between qRT-PCR and chip-based digital PCR platforms.

Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain (Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor (greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject's skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.

Rescue of Low-Yield DNA Samples for Next-Generation Sequencing Using Vacuum Centrifugal Concentration in a Clinical Workflow

The implementation of next-generation sequencing (NGS) in clinical oncology has enabled the analysis of multiple cancer-associated genes for diagnostics and treatment purposes. The detection of pathogenic and likely pathogenic mutations is crucial to manage the disease. Obtaining the mutational profile may be challenging in samples with low yields of DNA—reflected by the type of biological material, such as formalin-fixed paraffin-embedded tissue (FFPE), needle biopsies, and circulating free/tumor DNA, as well as a sparse tumor content. Moreover, standardized strict procedures for the extraction of DNA in a clinical setting might contribute to lower amounts of DNA per µL. The detection of variants in low-yield DNA samples remains a challenge in clinical diagnostics, where molecular analyses such as NGS are needed. Here, we performed vacuum centrifugation on DNA extracted from five FFPE tissue blocks, with concentrations below 0.2 ng/µL. Through NGS analysis, we found that low-yield DNA samples could be concentrated to sufficient levels, without compromising the mutational profile.

Starch treatment improves the salivary proteome for subject identification purposes

Identification of subjects, including perpetrators, is one of the most crucial goals of forensic science. Saliva is among the most common biological fluids found at crime scenes, containing identifiable components. DNA has been the most prominent identifier to date, but its analysis can be complex due to low DNA yields and issues preserving its integrity at the crime scene. Proteins are emerging as viable candidates for subject identification. Previous work has shown that the salivary proteome of the least-abundant proteins may be helpful for subject identification, but more optimized techniques are needed. Among them is removing the most abundant proteins, such as salivary α-amylase. Starch treatment of saliva samples elicited the removal of this enzyme and that of glycosylated, low-molecular-weight proteins, proteases, and immunoglobulins, resulting in a saliva proteome profile enriched with a subset of proteins, allowing a more reliable and nuanced subject identification.

DNA extraction protocol impacts ocular surface microbiome profile.

The aim of this study is to provide a reference frame to allow the comparison and interpretation of currently published studies on 16S ribosomal ribonucleic acid amplicon sequencing of ocular microbiome samples using different DNA extraction protocols. Alongside, the quantitative and qualitative yield and the reproducibility of different protocols has been assessed. Both eyes of 7 eligible volunteers were sampled. Five commercially available DNA extraction protocols were selected based on previous publications in the field of the ocular surface microbiome and 2 host DNA depletion protocols were added based on their reported effective host DNA depletion without significant reduction in bacterial DNA concentration. The V3-V4 region of the 16S rRNA gene was targeted using Illumina MiSeq sequencing. The DADA2 pipeline in R was used to perform the bio-informatic processing and taxonomical assignment was done using the SILVA v132 database. The Vegdist function was used to calculate Bray-Curtis distances and the Galaxy web application was used to identify potential metagenomic biomarkers via linear discriminant analysis Effect Size (LEfSe). The R package Decontam was applied to control for potential contaminants. Samples analysed with PowerSoil, RNeasy and NucleoSpin had the highest DNA yield. The host DNA depletion kits showed a very low microbial DNA yield; and these samples were pooled per kit before sequencing. Despite pooling, 1 of both failed to construct a library.Looking at the beta-diversity, clear microbial compositional differences - dependent on the extraction protocol used - were observed and remained present after decontamination. Eighteen genera were consistently retrieved from the ocular surface of every volunteer by all non-pooled extraction kits and a comprehensive list of differentially abundant bacteria per extraction method was generated using LefSe analysis. High-quality papers have been published in the field of the ocular surface microbiome but consensus on the importance of the extraction protocol used are lacking. Potential contaminants and discriminative genera per extraction protocol used, were introduced and a reference frame was built to facilitate both the interpretation of currently published papers and to ease future choice - making based on the research question at hand.

Homologous recombination deficiency in newly diagnosed FIGO stage III/IV high-grade epithelial ovarian cancer: a multi-national observational study

ObjectiveOlaparib plus bevacizumab maintenance therapy improves survival outcomes in women with newly diagnosed, advanced, high-grade ovarian cancer with a deficiency in homologous recombination. We report data from the first year...

Prenatal exome sequencing and impact on perinatal outcome: cohort study.

First, to determine the uptake of prenatal exome sequencing (pES) and the diagnostic yield of pathogenic (causative) variants in a UK tertiary fetal medicine unit following the introduction of the NHS England Rapid Exome Sequencing Service for fetal anomalies testing (R21 pathway). Second, to identify how the decision to proceed with pES and identification of a causative variant affect perinatal outcomes, specifically late termination of pregnancy (TOP) at or beyond 22 weeks' gestation. This was a retrospective cohort study of anomalous fetuses referred to the Liverpool Women's Hospital Fetal Medicine Unit between 1 March 2021 and 28 February 2022. pES was performed as part of the R21 pathway. Trio exome sequencing was performed using an Illumina next-generation sequencing platform assessing coding and splice regions of a panel of 974 prenatally relevant genes and 231 expert reviewed genes. Data on demographics, phenotype, pES result and perinatal outcome were extracted and compared. Descriptive statistics and the χ-square or Fisher's exact test were performed using IBM SPSS version 28.0.1.0. In total, 72 cases were identified and two-thirds of eligible women (n = 48) consented to trio pES. pES was not feasible in one case owing to a low DNA yield and, therefore, was performed in 47 cases. In one-third of cases (n = 24), pES was not proposed or agreed. In 58.3% (14/24) of these cases, this was because invasive testing was declined and, in 41.7% (10/24) of cases, women opted for testing and underwent chromosomal microarray analysis only. The diagnostic yield of pES was 23.4% (11/47). There was no overall difference in the proportion of women who decided to have late TOP in the group in which pES was agreed compared with the group in which pES was not proposed or agreed (25.0% (12/48) vs 25.0% (6/24); P = 1.0). However, the decision to have late TOP was significantly more frequent when a causative variant was detected compared with when pES was uninformative (63.6% (7/11) vs 13.9% (5/36); P < 0.0009). The median turnaround time for results was longer in cases in which a causative variant was identified than in those in which pES was uninformative (22 days (interquartile range (IQR), 19-34) days vs 14 days (IQR, 10-15 days); P < 0.0001). This study demonstrates the potential impact of identification of a causative variant by pES on decision to have late TOP. As the R21 pathway continues to evolve, we urge clinicians and policymakers to consider introducing earlier screening for anomalies, developing robust guidance for late TOP and ensuring optimized support for couples. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.

Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization. Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples. The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness. Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

A protocol specialized for microbial DNA extraction from living poplar wood

Microbial DNA extraction is a critical step in metagenomic research. High contents of chemical substances in wood tissues always cause low microbial DNA yield and quality. Up to date, almost no specialized methods involved in microbial DNA extraction from living wood were reported. In this study, an improved protocol (M1) concerning microbial DNA extraction from living poplar wood was developed. We compared microbial DNA yield and quality by M1 with those by other seven methods, including PowerSoil DNA isolation kit (M2), two soil microbial DNA extraction methods (M3 and M4), poplar genomic DNA extraction method from wood (M5), and microbial DNA extraction method from herb stems (M6), isolating bacteria (M7) and isolating fungus (M8). Results showed that M1 yielded much better quality and concentration of microbial DNA than the other methods (M2-M8) from both poplar wetwood and sapwood tissues. Following M1 protocol, 1 g of wetwood sample could yield 272.27 ng/ul (vol=50 ul) pure microbial DNA with the absorption ratios of 1.87 (A260/A230) and 1.66 (A260/A280). For 1 g of sapwood sample, these values were 361.83 ng/ul, 1.85 and 2.24, respectively. These DNA could be stably visualized by agarose gel electrophoresis and amplified by primer sets of bacteria (16S V3-V4, 16S-V4, 16S V4-V5) and fungus (ITS1, ITS2). While, the other seven methods only obtained less or contaminated microbial DNA, which could not be amplified stably by aforementioned primer sets. Our protocol provided an approach for microbial community study in living poplar wood in a more accurate way by molecular biology techniques.

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

Bovine milk mainly contains two types of β-casein: A1 and A2 variants. In recent years, a new variety of cows’ milk has emerged in dairy sector called “A2 milk”. This novel product is characterised by the absence of A1 β-casein, which has been associated with possible gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) release during gastrointestinal digestion. In this context, methods to verify A1 allele absence in A2 milk are required as a quality control in the A2 milk commercialisation. Therefore, the aim of the present study was to develop a locked nucleic acid (LNA) probe-based duplex real-time PCR (qPCR) assay for A1 allele detection in A2 milk samples. Firstly, four DNA isolation methods from milk somatic cells were optimised and evaluated. Results suggests that the commercial kit NucleoSpin Tissue was the most suitable method in terms of DNA quality and amplificability for downstream applications. Then, optimisation and validation of the qPCR assay were carried out. For both A1 and A2 alleles, the absolute limits of detection of this qPCR assay were 7.3 DNA copies/reaction (2 x 10 −5 ng DNA) and 30.4 DNA copies/reaction (0.1 ng DNA) at a 95% confidence level with synthetic reference DNA samples and heterozygous genotyped DNA sample, respectively. The relative limits of detection were 2% (15 copies) and 5% (152 copies) for A1 allele in A2 samples at a 95% confidence with synthetic reference and genotyped DNA samples, respectively. The qPCR assay was robust, with intra- and inter-assay variability below 4.3%, and specific, differentiating between A1 and A2 alleles with 100% genotyping accuracy. In conclusion, this cost-effective and fast method could be used to discriminate A1 allele in A2 samples and, consequently, to verify A1 allele absence in “A2 milk” by screening commercial product on the market. • The LNA duplex qPCR assay developed could be used to verify A1 allele absence in “A2 milk”. • LNA probes reliably discriminated between A1 and A2 alleles. • The relative limits of detection reach up to 2% (15 copies) for A1 allele in A2 samples at a 95% confidence. • Milk is a challenging matrix for DNA isolation due to the presence of PCR inhibitors that leads to low DNA yield.

Rapid Freezing of Skeletal and Cardiac Muscles Using Isopentane Cooled with Liquid Nitrogen and Tragacanth Gum for Histological, Genetic, and Protein Expression Studies.

Histological and molecular genetic evaluation of skeletal and cardiac muscles is an indispensable part of understanding muscle biology and the pathology of muscle disorders. Proper processing of the muscle tissue is a prerequisite for optimal evaluation. However, the processing of skeletal muscle samples often comes with many challenges. One of the commonly used methods of frozen tissue preparation involves optimal cutting temperature compound (OCT compound) embedding. This method is considered optimal for the processing of most of the routinely studied tissue samples. However, the processing of skeletal muscle samples using this method is often unsuitable as it causes artifacts and low DNA, RNA, and protein yield and quality due to the slow freezing of skeletal muscle tissues that allows ice crystals to form. One of the most suitable methods for skeletal muscle tissue processing for histological, genetic, and molecular studies is rapid freezing of freshly collected tissue samples using isopentane cooled with liquid nitrogen and tragacanth gum, which provides distinct advantages in consuming less time, preserving the cell morphology, and helping higher nucleic acids and protein yields. This chapter describes a protocol for rapid freezing of freshly collected skeletal muscle tissues using isopentane pre-chilled with liquid nitrogen and tragacanth gum. Skeletal muscle tissue samples processed using this protocol can be used for histological and immunological staining investigations and studies requiring DNA, RNA, and proteins from these tissues.

Optimization of bacterial DNA and endotoxin extraction from settled airborne dust

Collecting and obtaining sufficient amount of airborne particles for multiple microbial component assessments can be challenging. A passive dust sampling device, the electrostatic dust fall collector (EDC) has been established for assessing airborne exposures including endotoxin and glucans. Recently, with advances in next-generation sequencing techniques, EDCs were used to collect microbial cells for DNA sequencing analysis to promote the study of airborne bacterial and fungal communities. However, low DNA yields have been problematic when employing passive sampling with EDC. To address this challenge, we attempted to increase the efficiency of extraction. We compared DNA extraction efficiency of bacterial components from EDCs captured on filters through filtration using five extraction techniques. By measuring the abundance, diversity and structure of bacterial communities using qPCR and amplicon sequencing targeting 16S rRNA genes, we found that two techniques outperformed the rest. Furthermore, we developed protocols to simultaneously extract both DNA and endotoxin from a single EDC cloth. Our technique promotes a high quality to price ratio and may be employed in large epidemiological studies addressing airborne bacterial exposure where a large number of samples is needed.

Comparison of DNA preservation between adult and non-adult ancient skeletons.

Studies evaluating DNA preservation in non-adults, or comparing preservation in adults and non-adults, are very rare. This study compares the preservation of DNA in the skeletal remains of adults and non-adults. It compares the quality and quantity of DNA recovered from different skeletal elements of adults and non-adults, and from non-adults of different age classes. In addition, the preservation of DNA in males and females is compared. Bone DNA preservation was estimated by measuring nuclear DNA concentration and its degradation, and through STR typing success. The study analyzed 29 adult skeletons and 23 non-adult skeletons from the Ljubljana-Polje archeological site, dating from the seventeenth to nineteenth century, and up to four skeletal elements (petrous bone, femur, calcaneus, and talus) were included. After full demineralization extraction, the PowerQuant System and the PowerPlex ESI 17 Fast System (Promega) were used for qPCR and STR typing, respectively. The results showed that, among the four bone types analyzed, only the petrous bone proved to be a suitable source of DNA for STR typing of non-adult skeletal remains, and DNA yield is even higher than in the adult petrous bone, which can be attributed to the higher DNA degradation observed in the adult petrous bone. In adult skeletons, petrous bones and tali produced high STR amplification success and low DNA yield was observed in adult femurs. The results of this study are applicable for the sampling strategy in routine forensic genetics cases for solving identification cases, including badly preserved non-adult and also adult skeletons.

Organellar microcapture to extract nuclear and plastid DNA from recalcitrant wood specimens and trace evidence

BackgroundIllegal logging is a global crisis with significant environmental, economic, and social consequences. Efforts to combat it call for forensic methods to determine species identity, provenance, and individual identification of wood specimens throughout the forest products supply chain. DNA-based methodologies are the only tools with the potential to answer all three questions and the only ones that can be calibrated “non-destructively” by using leaves or other plant tissue and take advantage of publicly available DNA sequence databases. Despite the potential that DNA-based methods represent for wood forensics, low DNA yield from wood remains a limiting factor because, when compared to other plant tissues, wood has few living DNA-containing cells at functional maturity, it often has PCR-inhibiting extractives, and industrial processing of wood degrades DNA. To overcome these limitations, we developed a technique—organellar microcapture—to mechanically isolate intact nuclei and plastids from wood for subsequent DNA extraction, amplification, and sequencing.ResultsHere we demonstrate organellar microcapture wherein we remove individual nuclei from parenchyma cells in wood (fresh and aged) and leaves of Carya ovata and Tilia americana, amyloplasts from Carya wood, and chloroplasts from kale (Brassica sp.) leaf midribs. ITS (773 bp), ITS1 (350 bp), ITS2 (450 bp), and rbcL (620 bp) were amplified via polymerase chain reaction, sequenced, and heuristic searches against the NCBI database were used to confirm that recovered DNA corresponded to each taxon.ConclusionOrganellar microcapture, while too labor-intensive for routine extraction of many specimens, successfully recovered intact nuclei from wood samples collected more than sixty-five years ago, plastids from fresh sapwood and leaves, and presents great potential for DNA extraction from recalcitrant plant samples such as tissues rich in secondary metabolites, old specimens (archaeological, herbarium, and xylarium specimens), or trace evidence previously considered too small for analysis.

Conservation genomics and systematics of a near-extinct island radiation.

Conservation benefits from incorporating genomics to explore the impacts of population declines, inbreeding, loss of genetic variation and hybridization. Here we use the near-extinct Mariana Islands reedwarbler radiation to showcase how ancient DNA approaches can allow insights into the population dynamics of extinct species and threatened populations for which historical museum specimens or material with low DNA yield (e.g., scats, feathers) are the only sources for DNA. Despite their having paraphyletic mitochondrial DNA (mtDNA), nuclear single nucleotide polymorphisms (SNPs) support the distinctiveness of critically endangered Acrocephalus hiwae and the other three species in the radiation that went extinct between the 1960s and 1990s. Two extinct species, A. yamashinae and A. luscinius, were deeply divergent from each other and from a third less differentiated lineage containing A. hiwae and extinct A. nijoi. Both mtDNA and SNPs suggest that the two isolated populations of A. hiwae from Saipan and Alamagan Islands are sufficiently distinct to warrant subspecies recognition and separate conservation management. We detected no significant differences in genetic diversity or inbreeding between Saipan and Alamagan, nor strong signatures of geographical structuring within either island. However, the implications of possible signatures of inbreeding in both Saipan and Alamagan, and long-term population declines in A. hiwae that pre-date modern anthropogenic threats require further study with denser population sampling. Our study highlights the value that conservation genomics studies of island radiations have as windows onto the possible future for the world's biota as climate change and habitat destruction increasingly fragment their ranges and contribute to rapid declines in population abundances.

Comparability of CMV DNA Extraction Methods and Validation of Viral Load.

Human cytomegalovirus is a herpesvirus that has a worldwide seroprevalence of more than 60% of adults in developed countries and 90% in developing countries. Severe disabilities in newborns are characteristic of the human cytomegalovirus congenital infection, and this virus is implicated in graft rejection in transplant patients. To treat and follow-up the infection, the CMVPCR viral loads are required, and the DNA extraction step remains very important; however, the quantity, quality, and purity of extracted DNA from different biological fluids influence the results of PCR amplification, that is why for reliable results, the choice of nucleic acid extraction methods requires careful attention. Materials and methods: In this study, we compare 4 protocols, I (EZ1 DSP Virus kit), II (EZ1 Virus mini kit), III (QIAamp DSP virus kit), and IV (heating); the extractions are made from plasma collected on EDTA tubes, and the concentration of extracted DNA was measured on NanoDrop Lite followed by real-time CMVPCR using an Artus CMV QS-RGQ kit. All protocols are performed following the manufacturer’s instructions. Results: This study is conducted on the samples of 135 transplant patients whose follow-up medical tests related to human cytomegalovirus infection; since most of the CMVPCR results are negative, we have chosen the 10 CMVPCR positive samples and 2 negative samples as controls to conduct this comparison study. By using NanoDrop Lite to evaluate the DNA concentration, the yield of extracted DNA is higher in our heating protocol than other protocols, the EZ1 DSP virus kit and EZ1 Virus mini kit show homogeneous quantities, and the QIAamp DSP virus kit shows very low DNA yields. Comparing cycle threshold and viral loads by real-time PCR, all these protocols identified negative samples (100%), and the previously positive samples used were as follows: protocol IV (90%), protocol II (60%), and protocol I (40%). QIAamp DSP virus kit results were not real-time PCR applicable and were non-conclusive because of the low DNA yields. Conclusion: Our developed heating method (protocol IV) is very effective, reliable, simple, fast, and cheap compared to the other protocols in our study.

One size does not fit all: Tuning eDNA protocols for high‐ and low‐turbidity water sampling

AbstractFindings from eDNA metabarcoding are strongly influenced by experimental approach, yet the effect of pre‐PCR sample processing on taxon detection and estimates of biodiversity across different water types is still poorly resolved. To fill this data gap, we investigated the impact of sampling effort, extraction method, and filter pore size on DNA yield, PCR inhibition, and 16S rDNA metabarcoding results for fishes in water samples collected from inshore turbid‐ and offshore clear‐water environments. The turbid‐water samples had high concentrations of suspended organic and/or inorganic material and yielded ~3.2× more DNA and exhibited high levels of PCR inhibition compared with the low‐turbidity, clear‐water samples. Importantly, there were no striking differences in the results of our metabarcoding experiments based on extraction method or filter pore size. While a small number of unique species of relatively low read count were detected in all turbid‐water treatments, most species were consistently detected across samples. Results for the clear‐water samples were strikingly different, with low DNA yield, high levels of variation across replicates, and a high number of non‐overlapping species across treatments. These findings indicate a patchy distribution of eDNA in offshore environments, which means higher volumes of water (≥ 2 L per replicate) must be filtered in habitats where target DNA is likely to be sparse. In semi‐closed systems such as estuaries, higher concentrations of target DNA are expected, and we found that either a 1.0 or 3.0 µm filter pore size was sufficient to capture standing diversity, while decreasing the risk of clogging. For economical DNA extraction and inhibitor removal, we recommend a combination of Omega Bio‐tek E.Z.N.A Tissue DNA kit followed by a PCR inhibitor removal step using the Zymo Kit. Finally, we emphasize that pilot studies should be undertaken whenever sampling in a new environment to identify which protocol is most appropriate.

Abstract 580: The broad surveillance and unbiased detection of circulating tumor DNA using next-generation sequencing without a priori knowledge of somatic mutations is a prognostically significant biomarker in pancreatic ductal adenocarcinoma

Abstract Background. Detection of low frequency tumor-derived variants in circulating cell-free DNA (ccfDNA; i.e., circulating tumor DNA, ctDNA) without a priori knowledge of mutations in the matched solid tumor is confounded by next-generation sequencing (NGS) noise and constrained by theoretical and practical limitations associated with low DNA yield from plasma. Here, we leverage technical replicates (two libraries prepared and sequenced independently from each extracted ccfDNA sample) to simultaneously suppress NGS-associated errors and improve detection of ctDNA derived from pancreatic ductal adenocarcinomas (PDACs). Methods. Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III) undergoing surgery aimed at complete total resection. For 11 patients, ccfDNA was also collected post-operatively within 100 days of surgery. Two technical replicates of ccfDNA libraries using molecular barcodes were independently generated using 2 mL plasma equivalents of ccfDNA for each library. Subsequently, libraries were capture-enriched using a 118 gene panel (~124 kb) and underwent NGS (paired-end, 125x2; HiSeq 2500). Consensus sequence determination and position-specific error modeling were coupled with restricting variant identification to nonreference alleles present in both patient replicates that corresponded to COSMIC-reported pathogenic mutations in PDAC. Results. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients (35.7%) with a variant allele frequency ranging from 0.08% to 1.59% (median: 0.25%). Of the nine ctDNA variants identified, five (55.6%) had a corresponding mutation in tumor DNA. The estimated median overall survival in patients with and without detection of pre-operative ctDNA was significantly different (312 vs. 826 days, respectively; χ2=5.3, P=0.0207). Using mutations identified in tumor tissue DNA to guide detection of pre-operative ctDNA yielded a similar survival analysis (369 vs 1,011 days, respectively; χ2=5.6, P=0.018). Detection of ctDNA in post-operative ccfDNA with or without guidance from tumor tissue mutations was not associated with outcomes. Applying the noise reduction strategy used to detect ctDNA in PDAC patients eliminated false positives from healthy controls. Conclusions. Acquisition of NGS data in replicate coupled with in silico error suppression strategies enabled detection of ctDNA without a priori knowledge of tumor variants, which overcomes a major obstacle limiting ccfDNA applications. Pre-operative ctDNA detected during a broad and untargeted survey of ccfDNA with NGS is a non-invasive, prognostic biomarker to help guide PDAC patient management. Citation Format: Kajsa E. Affolter, Sabine Hellwig, David A. Nix, Mary P. Bronner, Alun Thomas, Carrie L. Fuertes, Cindy L. Hamil, Ignacio Garrido-Laguna, Courtney L. Scaife, Sean J. Mulvihill, Hunter R. Underhill. The broad surveillance and unbiased detection of circulating tumor DNA using next-generation sequencing without a priori knowledge of somatic mutations is a prognostically significant biomarker in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 580.