Quality Of DNA Research Articles

Variations in antimicrobial resistance genes present in the rectal faeces of seals in Scottish and Liverpool Bay coastal waters

Antibiotic resistance genes originating from human activity are considered important environmental pollutants. Wildlife species can act as sentinels for coastal environmental contamination and in this study we used qPCR array technology to investigate the variety and abundance of antimicrobial resistance genes (ARGs), mobile genetic elements (MGEs) and integrons circulating within seal populations both near to and far from large human populations located around the Scottish and northwest English coast. Rectal swabs were taken from 50 live grey seals and nine live harbour seals. Nucleic acids were stabilised upon collection, enabling extraction of sufficient quality and quantity DNA for downstream analysis. 78 ARG targets, including genes of clinical significance, four MGE targets and three integron targets were used to monitor genes within 22 sample pools. 30 ARGs were detected, as well as the integrons intl1 and intl2 and tnpA transposase. Four β-lactam, nine tetracycline, two phenicol, one trimethoprim, three aminoglycoside and ten multidrug resistance genes were detected as well as mcr-1 which confers resistance to colistin, an important drug of last resort. No sulphonamide, vancomycin, macrolide, lincosamide or streptogramin B (MLSB) resistance genes were detected. Resistance genes were detected in all sites but the highest number of ARGs (n = 29) was detected in samples derived from grey seals on the Isle of May, Scotland during the breeding season, and these genes also had the highest average abundance in relation to the 16S rRNA gene. This pilot study demonstrates the effectiveness of a culture-independent workflow for global analysis of ARGs within the microbiota of live, free-ranging, wild animals from habitats close to and remote from human habitation, and highlights seals as a valuable indicator species for monitoring the presence, abundance and land-sea transference of resistance genes within and between ecosystems.

Mitochondrial genome data provide insights into the phylogenetic relationships within Triplophysadalaica (Kessler, 1876) (Cypriniformes, Nemacheilidae).

Due to the detrimental effect of formaldehyde on DNA, ethanol has replaced formalin as the primary preservative for animal specimens. However, short-term formalin fixation of specimens might be applied during field collection. In an increasing number of studies, DNA extraction and sequencing have been successfully conducted from formalin-fixed specimens. Here the DNA from five specimens of Triplophysadalaica (Kessler, 1876) were extracted and performed high-throughput sequencing. Four of the specimens underwent short-term fixation with formalin and were subsequently transferred to ethanol. One was continuously stored in ethanol. No significant difference of DNA quality and amount were observed among these samples. Followed by assembly and annotation, five mitochondrial genomes ranging in length from 16,569 to 16,572 bp were obtained. Additionally, previously published data of other individuals or species were included to perform phylogenetic analyses. In the reconstructed trees, all eight individuals of T.dalaica form a monophyletic group within the Triplophysa branch. The group is divided into three clades: (1) samples from the Yellow River, (2) those from the Yangtze River, and (3) those from the Haihe River, and the Lake Dali Nur. This study sheds initial light on the phylogeographic relationships among different populations of T.dalaica, and will support the research about its evolutionary history in the future.

Optimization of the protocol for the assembly of recombinant adenoassociated serotype 2 viruses for the delivery of African swine fever virus genes into mammalian cells

African swine fever (ASF) is a highly contagious viral disease of the Suidae family representatives, the mortality rate in primary foci of which reaches 100 %. To date, no specific means of preventing ASF have been developed. Despite the fact that researchers have proposed various methods for creating candidate vaccines against ASF, the issue of developing alternative antigenic variants with low reactogenicity and high immunogenicity is still relevant. It is known that the production of recombinant adeno-associated virus, a potential tool for delivering ASF virus target genes into mammalian cells, is influenced by many factors, in particular, the cell line, expression system, cell culture conditions after transfection, and the quality of the initial plasmid DNA. This work presents the results of optimization of the assembly protocol for recombinant AAV2 carrying the major capsid protein gene of the ASFV B646L as a model cargo. During the research, it was established that the protocol used allows to achieve a veritable virus titer of (2.45 ± 0.17) × 107 viral particles per μl, while the share of fully assembled viral capsids accounts for up to (79.3 ± 2.3) % of all genomic copies. When assessing the potential cytopathogenic effect of recombinant AAV2 on target cells (SPEV, porcine MSCs), it was found that high MOI (up to 10,000 viral particles per cell) does not lead to an increase in the proportion of apoptotic cells. The functionality of the developed AAV2-based construct was confirmed: in the lysates of transduced cells, the mature p72 protein with a molecular weight of 73 kDa was detected, specifically reacting in a western blot with hyperimmune pig serum. Our data confirm the potential of AAV2 as a tool for delivering ASF virus genes into porcine cells, which makes it a promising basis for the design of candidate vaccines.

CTAB DNA Extraction Method Optimization for Quantification of Lard Content in Cheese and Butter Using Real-Time PCR

ABSTRACT Porcine adulteration in dairy products for economic purposes has been tackled mostly by protein- and lipid-based porcine detection methods. In this study, a DNA-based approach was used to determine the presence of porcine DNA in dairy products, especially butter and cheese. DNA was extracted by optimizing the conventional CTAB extraction method followed by an assessment of DNA fragmentation by real-time PCR assays targeting different sizes of amplicon. Butter and cheese DNA samples were analyzed using both endogenous and porcine-specific real-time PCR assays and the lard content was estimated based on the linear regression analysis of a series of different percentages of lard-adulterated butter and cheese DNA samples. Even though the optimization steps did not improve the DNA yield, the successful amplification of the 200 bp amplicon depicted high DNA quality. Approximately 1% lard was able to be quantified exhibiting high potential of the assay in quantifying porcine content in lard-adulterated highly processed food products.

The Effects on Post-Thaw Sperm Quality and Nuclear DNA Integrity of Supplementation of Low-Density Lipoprotein to Freezing Extender in the Mouse

Mice are an important research tool for genetic and molecular biology, allowing researchers to explore a variety of human illness models. Egg yolk is a common component of semen extenders for domestic animals and low-density lipoproteins (LDL) from egg yolk have some cryoprotective properties. This study aimed to investigate sperm quality characteristics and nuclear DNA integrity after post-thawing in an extender (18% raffinose + 3% skim milk) supplemented with different concentrations of LDL (2.5%, 5.0%, 7.5%, or 10%) in mice. 18% Raffinose+3% skim milk extender was used as a control group without LDL. CD-1 mice were used in the study, and semen was collected from the cauda epididymis and diluted with the extender. The straws were then frozen and thawed to evaluate progressive motility, viability, plasma membrane (HOST), acrosome, and nuclear DNA integrity parameters. Fresh sperm had the highest progressive motility, viability, plasma membrane integrity, and longevity (endurance) of progressive motility for 4 h in HTF solution. The greatest spermatologic results, including nuclear DNA integrity, were determined in fresh sperm (p

Using the DNA Integrity Number to Analyze DNA Quality in Specimens Collected from Liquid-Based Cytology after Fine-Needle Aspiration of Breast Tumors and Lesions

Introduction: Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage. Methods: We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine-needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA integrity number (DIN) score analysis. Results: Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens. Conclusion: Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis.

Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.

Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cancer (nNGM). Four centers applied the anchored multiplex ArcherDX-Variantplex nNGMv2 panel to re-analyze samples pre-tested during routine diagnostics. Data analyses were performed by each center and compiled centrally according to study design. Pre-defined standards were utilized, and panel sensitivity was determined by dilution experiments. nNGMv2 panel sequencing was successful in 98.9% of the samples (N = 90). With default filter settings, all but two potential MET exon 14 skipping variants were identified at similar allele frequencies. Both MET variants were found with an adapted calling filter. Three additional variants (KEAP1, STK11, TP53) were called that were not identified in pre-testing analyses. Only total DNA amount but not a qPCR-based DNA quality score correlated with average coverage. Analysis was successful with a DNA input as low as 6.25ng. Anchored multiplex PCR-based sequencing (nNGMv2) and a sophisticated user-friendly Archer-Analysis pipeline is a robust and specific technology to detect tumor genetic mutations for precision medicine of lung cancer patients.

DETERMINING GOOD DNA QUALITY OF FFPE CRC TISSUES VIA A MACRODISSECTION-DNA EXTRACTION PROTOCOL

A good DNA quality and quantity from a formalin-fixed paraffin-embedded (FFPE) tissue sample is crucial for a better downstream genomic analysis. Previous research has focused on comparing FFPE DNA extraction kits, but the improvement in kit outcome has yet to be studied. This study aimed to make some modifications to a selected Qiagen DNA extraction manual protocol using a manual macrodissected FFPE colorectal cancer (CRC) tissue sample. A variety of DNA extraction protocol-related variables, such as washing steps, tissue sections, and FFPE tissue age have been investigated to determine the DNA quality and quantity of the FFPE tissues. The prechilled absolute ethanol washing step showed the highest DNA concentration with good purity ratios. One, two, or four tissue sections using the washing step were sufficient to obtain adequate DNA concentrations with acceptable DNA purity ratios. 1% or 2% agarose gel electrophoresis with the pre-casting method allowed better visualisation of FFPE DNA. Based on the ideal quality and quantity, we chose a manual macrodissected DNA extraction protocol employing 4 x 10 µm FFPE tissue sections generated in recent years with an optimised prechilled absolute ethanol washing step.

Milk Protein Genes Polymorphism in Indigenous and Crossbred Cattle from a private Dairy Farm in Ethiopia

Kappa casein and beta lactoglobulin genes are major milk proteins which have a direct effect on protein content in dairy cattle. Molecular-based selection through the identification of genetic polymorphism of major protein genes can be used to gain genetic improvement of milk protein yield. The objective of this study was to identify kappa casein (CSN3) and beta lactoglobulin (LGB) genes polymorphisms in indigenous and crossbreed cattle. A total of 90 whole blood samples were collected from individual animal in a private dairy farm. DNA extraction and quality assessment were done salting out procedure and gel electrophoresis, respectively. Polymerase chain reaction (PCR) was performed with gene specific primers. For genotyping, PCR products of CSN3 gene was digested with HinfI and HindIII while LGB gene was digested with HaeIII restriction enzymes. Two haplotypes A and B; three genotypes, AA, AB and BB were observed at CSN3 of HinfI site and LGB HaeIII site, but only AA and AB were observed at CSN3 of HindIII site in crossbred and indigenous cattle populations. However, at CSN3 locus, A allele was found to be more common (0.65) in indigenous cattle than the B allele (0.35), while B (0.51) allele and AB genotype (0.81) were more frequent in crossbred cattle. But, LGB B allele was higher in indigenous cattle (0.67) compared to Allele A (0.33). LGB BB genotype (0.57) is higher followed by AA genotype in indigenous cattle while both allele and genotypic frequencies are equal in crossbred cattle. Both CSN3 and LGB loci were polymorphic in studied populations. Expected heterozygosity was higher in crossbred (0.49, 0.50) than in indigenous (0.38, 0.33) cattle at CSN3 and LGB locus, respectively which might be due to breed variation. The current findings showed that both CSN3 and LGB genes could be promising diagnostic markers in selecting dairy cattle breed. However, further investigations with large sample size and association study with milk composition is required to substantiate the current result.

Beneficial effects of oral antioxidant supplementation on semen quality parameters, reproductive hormones, and sperm DNA integrity in men with idiopathic oligoasthenoteratozoospermia.

Recently, oral antioxidants in combined forms have been used to treat men with idiopathic infertility. This study aimed to evaluate the effects of treatment with vitamin C, vitamin E, selenium, zinc, arginine, L-carnitine, and coenzyme Q10 on sperm quality parameters, DNA integrity, reproductive hormones, and pregnancy rates in men with infertility and idiopathic oligoasthenoteratozoospermia (OAT). A prospective study was conducted on 420 men with infertility and idiopathic OAT who took an oral supplement of antioxidant SP-Power tablets twice daily for 6 months. Semen quality, reproductive hormones, and the DNA fragmentation index (DFI) were evaluated at baseline and at 3 and 6 months after supplementation, using the World Health Organization 2021 guidelines. No significant difference was observed in volume or the percentage of typical morphology during treatment. A significant improvement in sperm concentration was observed after supplementation (8.67±1.41, 12.17±1.91, and 19.01±0.86 at baseline, 3, and 6 months respectively, p<0.01). The total motility, progressive motility, and total motile sperm count also increased significantly (p<0.01), whereas the DFI decreased after 6 months. There was an increase in normal FSH levels and testosterone levels after 6 months of supplementation of antioxidant SP-Power but these differences were not statistically significant (p=not significant and p=0.06, respectively). Supplementation with SP-Power tablets improved sperm quality parameters, sperm DFI, some reproductive hormones, and pregnancy rates in men with infertility and idiopathic OAT, which could be attributed to the supplement's synergistic antioxidant action. Further studies are needed to determine the effects of supplementation on oxidative stress markers.

Abstract 4957: DNA quality control metrics in cryopreserved tissue outperform DNA extracted from FFPE tissue

Abstract Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing of tumor tissue in clinical cancer care comes primarily from formalin-fixed, paraffin-embedded tissue (FFPE). Specific to genomics assays, numerous studies have shown significant discordance in genetic information obtained from FFPE samples and frozen samples. To explain the discordance between FFPE samples and cryopreserved samples, a head-to-head comparison between FFPE and cryopreserved tissues was performed to analyze the DNA yield, DNA purity and DNA quality in terms of DNA length. Additionally, short-read sequencing was performed on matched samples and quality control metrics were compared between the two sample types. Matched human tumors (n = 50) were processed either by formalin fixation and paraffin embedding or placed in cryovials containing cryopreservation medium. These cryovials were cooled slowly to -80°C and stored in liquid nitrogen. DNA was extracted using the same protocol for both tissue types with the exception that tissues embedded in paraffin were first dewaxed followed by a multistep rehydration protocol. Samples were weighed and calibrated for mass. After the column purification and elution, sample concentration and purity were measured. DNA fragment length was measured on a fragment microelectrophoresis analyzer. Graded amounts of tumor tissue were used to determine the lowest starting material needed to extract 200 ng of DNA. The average of all the samples reached the minimal threshold of 200 ng. However,74% of FFPE specimens failed to meet the minimum 40 ng/mg, whereas only 21% were below the threshold in the cryopreserved samples. In the cryopreserved group, the average DNA yield was 222.1 ng/mg, whereas 52.8 ng/mg was obtained from FFPE tissue. For DNA purity in cryopreserved tissues, the 260/280 ratio was 1.76, and in FFPE tissues the mean was 1.78. The DNA Quality Number (DQN) is a measurement of DNA fragment length and the percentage that exceeds the threshold of 300 bp. For FFPE, the DQN was 4.4 compared to a DQN of 9.8 for the cryopreserved samples. Setting a higher threshold of DNA length to 40,000 bp and measuring the area under the curve (AUC), it was observed that cryopreserved samples were 9-fold higher in fragments greater than 40,000 bp. Cryopreserved cancer tissue provides superior quality assurance measurements of DNA over FFFPE. Treatment decisions based on molecular results demand accuracy and validity. The pathology community should support efforts to cryopreserve cancer biospecimens in the clinical setting to provide valid molecular testing results. The automatic pickling of tumor specimens in formalin is no longer an acceptable default. Citation Format: Jared James Barrott, Nikole O'Neal, Jeffrey Okojie, David Booker, Ken Dixon. DNA quality control metrics in cryopreserved tissue outperform DNA extracted from FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4957.

Abstract 978: Functional characterization and a real-world clinical laboratory pilot of the Foundation for the National Institutes of Health’s (FNIH) circulating tumor DNA (ctDNA) quality control materials (QCMs)

Abstract Background: Analysis of ctDNA now permits assessment of targetable mutations (e.g. EGFR L858R/Ex19del) using noninvasive blood tests. There are several FDA-approved tests using digital PCR (ddPCR) or next-generation sequencing (NGS) and many more laboratory developed tests in use. The FNIH Biomarkers Consortium identified the absence of validated standards to qualify the performance of ctDNA assays as a critical need and set out to aid in the characterization of QCMs. Here we report on a commutability study comparing the performance of three commercial QCMs to clinical samples at or above LoD (limit of detection) and update on a real-world assessment of these QCMs by 11 clinical laboratories spanning 4 continents and 3 assay modalities. Methods: QCMs were provided by Horizon Discovery (H), Thermo Fisher (T) and SeraCare (S). Clinical samples with known EGFR L858R and Ex19del mutations were prepared by diluting specimens at known concentrations into cell-free DNA (cfDNA) from healthy donors. The functional characterization compared detection of the mutations in QCM to clinical samples and consisted of 2 parts: (1) Evaluation of EGFR L858R/Ex19del (range 0.1% to 5% allele frequency [AF]) by ddPCR, amplicon and hybrid-capture NGS; (2) Evaluation near the LoD of the amplicon NGS assay at the assay LoD95 of 0.3% AF. For the clinical pilot, participants were sent QCM pools formulated at 0.5% and 1% AF by ddPCR. Some participants also tested clinical specimens with EGFR L858R and Ex19del at 0.5% and 1% AF by ddPCR. All participants were blinded to AF and identity of the manufacturer. Results: For Part 1, QCM results from hybrid-capture NGS of EGFR L858R were similar to ddPCR; for Ex19del, QCM AF trended lower compared to ddPCR. In contrast, L858R clinical samples showed a strong positive bias compared to ddPCR. For amplicon NGS, QCMs performed similarly to clinical samples, with amplicon NGS AF closest to ddPCR AF for T QCM. For Part 2, the observed hit rates mostly approximated expected values. For H QCM, observed LoD95 (10% for EGFR L858R; 50% for Ex19del) and subsequent dilution hit rates were lower than expected. For the clinical pilot, median Ex19del AF of amplicon NGS were higher than for hybrid-capture NGS for both the 1% QCM formulations and 0.5% QCM formulations. Median QCM L858R AF were similar for amplicon and hybrid-capture NGS, with greatest contributor to variance being inter-lab differences. Conclusions: This project, unprecedented in the number of participants and analytical rigor, revealed unexpected differences in performance of both assays and QCMs. These findings merit consideration by laboratories that rely on QCMs to develop and perform assays for ctDNA testing as diagnostics. Citation Format: Cloud P. Paweletz, Thomas D. Forbes, Grace A. Heavey, Laura M. Yee, Hua-Jun He, Zhiyong He, Dana E. Connors, Daniel Stetson, Susan Keating, Kenneth D. Cole, Gary Kelloff, Robert T. McCormack, Mickey P. Williams, Chris Karlovich. Functional characterization and a real-world clinical laboratory pilot of the Foundation for the National Institutes of Health’s (FNIH) circulating tumor DNA (ctDNA) quality control materials (QCMs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 978.

Abstract 2926: The effect of cancer research autopsy parameters on DNA and RNA sequencing quality

Abstract Background: Cancer research autopsy genomic studies offer insight into the metastatic cancer landscape but come with complexities that relate to the sampling and processing of post-mortem tissue. Clarifying the effect of autopsy variables on pre- and post-sequencing quality control (QC) is an unmet need that may inform tissue collection strategies. Methods: The effect of age, sex, post-mortem interval (PMI), and sample type (primary, metastatic, or normal) on pre-sequencing QC (nucleic acid concentration and integrity) was examined in 2678 samples (88.6% metastatic, 8.0% primary, 3.4% normal) from 83 patients with melanoma, lung, renal, or prostate cancer in the PEACE study. In the lung cohort, 160 surgical samples were also included through the TRACERx study, allowing surgery-autopsy tissue comparisons. Post-sequencing QC metrics were evaluated for lung samples that underwent DNA (n=522) or RNA (n=366) sequencing. Results: RNA concentration and RIN were greater in surgical samples than those collected at autopsy. Across cohorts, metastatic autopsy samples had greater nucleic acid concentrations than primary or normal autopsy samples, but not integrity. DNA and RNA concentration and integrity differed significantly between primary tumor types. When comparing samples of different metastatic sites from the whole cohort, concentration was lowest in bone (DNA) or the digestive tract (RNA), while integrity was greatest in the brain and lowest in the digestive tract (DIN, RIN). Although autopsy variables like age, sex and PMI correlated with pre-sequencing QC metrics in univariate analysis, they were not found to significantly correlate with these metrics in multivariate analysis, which identified that only primary cancer type and metastatic site were independent determinants of concentration and integrity. Similarly, for post-DNA (whole exome) sequencing QC, only the metastatic site was found to independently influence sequencing QC metrics like total number of sequences, average sequence length, and FastQC score. For RNA sequencing, only the metastatic site was found to influence sequencing QC metrics like total number of sequences, percentage of non-duplicated sequences, one hit-one genome percentage, and the alignment percentage on the human genome. Discussion: The lack of influence of PMI on QC in the largest QC-focused autopsy cancer study to date suggests that quality tissue can be obtained from non-rapid autopsy programs, which are more feasible and less resource-intensive than rapid programs. Citation Format: Petros Fessas, Sonya Hessey, Corentin Richard, Cristina Naceur-Lombardelli, Sophia Ward, David A. Moore, Karolina Nowakowska, Blanca Trujillo, Irene Lobon, Scott T. Shepherd, Fiona Byrne, Samra Turajlic, Gerhardt Attard, Charles Swanton, Mariam Jamal-Hanjani. The effect of cancer research autopsy parameters on DNA and RNA sequencing quality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2926.

Pathology Laboratory Archives: Conservation Quality of Nucleic Acids and Proteins for NSCLC Molecular Testing.

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids' quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6-8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6-8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond.

Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy

Abstract Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions. Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay. Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models. Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels. Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7248.

Abstract 326: Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method

Abstract In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient surgical samples, where in most cases high quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and highly variable damage from fixation, storage, and extraction methods. It is difficult to obtain libraries with sufficient coverage and the sequencing artifacts arising from damaged DNA bases confound somatic variant detection. Additionally, many laboratories process FFPE tumor samples alongside matched, high quality, normal DNA and many library prep workflows are not readily compatible with both sample types. We developed a novel NGS library prep method compatible with both high quality and very low quality FFPE DNA samples, employing a novel enzymatic DNA repair mix, enzymatic fragmentation mix, and PCR master mix. To validate this workflow on real patient sample sets, we obtained DNA extracted from matched tumor and normal tissue of various tissue types preserved by both fresh frozen and FFPE methods. The FFPE DNA samples ranged in quality from DNA integrity number (DIN) 1.5 to 6.8, including samples stored for nearly 3 decades. We prepared libraries using this method compared against other library prep workflows and sequenced them by WGS and target capture. We assessed the performance of these workflows by library yield, library quality metrics, depth and evenness of coverage, and somatic variant calling with fresh frozen-extracted DNA providing the gold standard for library quality and mutation content. This new enzymatic fragmentation-based library prep workflow not only reduced the false positive rate in somatic variant detection by repairing damage-derived mutations in FFPE DNA samples but also improved the library yield, library quality metrics (including mapping, chimeras, and properly-paired reads), library complexity, coverage depth and uniformity, and hybrid capture library quality metrics. Comparing the variant calls from matched FFPE and frozen tissues revealed an improved sensitivity and accuracy of variant calling using this library prep method compared to mechanical shearing and other enzymatic fragmentation library prep approaches. This new suite of enzyme mixes allows even highly damaged FFPE samples to achieve high quality libraries with a greater sensitivity for somatic variant identification. The workflow is robust and flexible, compatible with both FFPE DNA and matched high quality DNA samples as well as automation-friendly for convenience in sample processing. Citation Format: Margaret R. Heider, Jian Sun, Brittany Sexton, Bradley W. Langhorst, Laura Blum, Pingfang Liu. Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 326.

Quantitative and qualitative analysis of three DNA extraction methods from soybean, maize, and canola oils and investigation of the presence of genetically modified organisms (GMOs)

The objective of this study was to develop a DNA-based method for the identification and tracking of edible oils, which is important for health management. Three different DNA extraction methods (CTAB, MBST kit, and manual hexane-based method) were used to obtain high-purity DNA from crude and refined soybean, maize, and canola oils. PCR was then conducted using specific primers to identify the presence of genes related to each oil type and to assess transgenicity. The results showed that DNA was present in crude and refined oils, but in very low amounts. However, using method 3 for DNA extraction provided sufficient quantity and quality of DNA for successful PCR amplification. The study concluded that the main challenge in DNA extraction from oils is the presence of PCR inhibitors, which can be overcome using the manual hexane-based method. Also, the examination of protein presence in the oils using SDS-PAGE did not indicate any protein bands.

Nanoparticle-Mediated Plasmid Curing in Combating Antibiotic Resistance in Pathogenic Bacteria

Background: Nanotechnology has emerged as a pivotal domain in material science research with extensive applications across various sectors including biotechnology and medicine. Nanoparticles offer unique properties facilitating advancements in nanobiotechnology, particularly in nanomedicine, to combat bacterial infections and antibiotic resistance. This study aimed to determine the application of nanoparticles, specifically nano-TiO2, in treating plasmid-mediated antibiotic resistance in both Gram-negative and Gram-positive bacteria. Method: We evaluated antibiotic and nanomaterial sensitivity through disc diffusion and broth microdilution assays. Plasmid curing experiments were conducted using varying concentrations of nano-TiO2 and SDS as curing agents, followed by plasmid isolation and DNA extraction. The efficacy of nano-TiO2 in plasmid curing and DNA extraction was assessed, alongside the impact on bacterial growth and antibiotic resistance. Results: Results showed successful plasmid elimination with nano-TiO2 treatment, evidenced by the loss of plasmid DNA bands. Additionally, nano-TiO2 substantially enhanced DNA extraction efficiency and quality. The study indicated nano-TiO2's potential in combating antibiotic resistance by targeting plasmids, thereby presenting a novel approach in molecular biology techniques. Conclusion: In conclusion, this study underscores the promising role of nanoparticles in addressing bacterial infections and combating antibiotic resistance. Nano-TiO2 emerges as a valuable tool in DNA purification and plasmid curing, offering new avenues in molecular biology and antibiotic resistance research. However, further investigations are warranted to elucidate the broader implications of nanoparticles across diverse bacterial species and strains. These findings represent a significant step towards harnessing the potential of nanotechnology in combating antimicrobial resistance and advancing healthcare paradigms.

New metagenomic procedure for the investigation of the eukaryotes present in the digestive gland of Mytillus galloprovincialis

Mussels’ aquaculture is one of the most important cultures in Europe, with a high level of self-sufficiency. In Spain, the main cultivated species is M. galloprovincialis, whose production takes place primarily in Galicia. In that region, a Protected Designation of Origin certification exists to certify the high quality of the product and its sustainability. This species is also present in other regions making the control of its traceability complicated. In order to distinguish Galician mussels from the others, the eukaryotes present in its digestive gland could be a useful tool. However, to use them as biomarkers, having an adapted protocol to recover the diversity present in the digestive gland is essential. In this work, we have selected a new protocol to optimize the study of the diversity of eukaryotes associated with the digestive gland of M. galloprovincialis. This protocol is based on a combination of a pre-treatment and a commercial DNA extraction kit, the amplification of DNA with two newly designed primer sets to avoid the DNA from the mussel, and the use of next-generation sequencing. The results showed a significant reduction of mussel DNA amplification and enough DNA quality for the subsequent sequencing, increasing the number of sequences recovered from other eukaryotes. This new protocol offers then a good chance to determine the diversity of eukaryotes present in the digestive gland of M. galloprovincialis, and it can be used in future studies to examinate the seasonal variation of the present species and to evaluate its potential as geographical traceability tool.

Testing of the Effect of Food Additives on the DNA Isolation from Mackerel and Fish Products

Authentication of fish products by DNA analysis requires the extraction of high quality DNA without the presence of inhibitors. Many nucleic acid isolation methods are currently available; silicate centrifugal columns have become very popular due to their speed and ease of extraction. However, their disadvantage may be the principle based on DNA charge, which may be affected by food composition, or clogging due to a poor sample pretreatment. The aim of this work was to compare three DNA isolation methods using different principles (silicate centrifugal columns, modified magnetic beads, Cetrimonium bromide and chloroform extraction) and to evaluate their suitability for DNA isolation from fish muscle. The criteria assessed were the recovery, purity and amplifiability of the isolated DNA. Mackerel tissue was analysed without and with the addition of additives commonly used in the manufacture of fish products, namely diphosphates (E 450) and colorants (E 110 and E 124), and the selected method was subsequently applied to commercial fish products. The modified method using the detergent CTAB proved to be the most suitable.