Fragment Length DNA Research Articles

Simulation of Radiation-Induced DNA Damage and Protection by Histones Using the Code RITRACKS.

(1) Background: DNA damage is of great importance in the understanding of the effects of ionizing radiation. Various types of DNA damage can result from exposure to ionizing radiation, with clustered types considered the most important for radiobiological effects. (2) Methods: The code RITRACKS (Relativistic Ion Tracks), a program that simulates stochastic radiation track structures, was used to simulate DNA damage by photons and ions spanning a broad range of linear energy transfer (LET) values. To perform these simulations, the transport code was modified to include cross sections for the interactions of ions or electrons with DNA and amino acids for ionizations, dissociative electron attachment, and elastic collisions. The radiochemistry simulations were performed using a step-by-step algorithm that follows the evolution of all particles in time, including reactions between radicals and DNA structures and amino acids. Furthermore, detailed DNA damage events, such as base pair positions, DNA fragment lengths, and fragment yields, were recorded. (3) Results: We report simulation results using photons and the ions 1H+, 4He2+, 12C6+, 16O8+, and 56Fe26+ at various energies, covering LET values from 0.3 to 164 keV/µm, and performed a comparison with other codes and experimental results. The results show evidence of DNA protection from damage at its points of contacts with histone proteins. (4) Conclusions: RITRACKS can provide a framework for studying DNA damage from a variety of ionizing radiation sources with detailed representations of DNA at the atomic scale, DNA-associated proteins, and resulting DNA damage events and statistics, enabling a broader range of future comparisons with experiments such as those based on DNA sequencing.

UV damage induces production of mitochondrial DNA fragments with specific length profiles.

UV light is a potent mutagen that induces bulky DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). Photodamage and other bulky lesions occurring in nuclear genomes can be repaired through nucleotide excision repair (NER), where incisions on both sides of a damaged site precede the removal of a single-stranded oligonucleotide containing the damage. Mitochondrial genomes (mtDNAs) are also susceptible to damage from UV light, but current evidence suggests that the only way to eliminate bulky mtDNA damage is through mtDNA degradation. Damage-containing oligonucleotides excised during NER can be captured with antidamage antibodies and sequenced (XR-seq) to produce high-resolution maps of active repair locations following UV exposure. We analyzed previously published datasets from Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster to identify reads originating from the mtDNA (and plastid genome in A. thaliana). In A. thaliana and S. cerevisiae, the mtDNA-mapping reads have unique length distributions compared to the nuclear-mapping reads. The dominant fragment size was 26 nt in S. cerevisiae and 28 nt in A. thaliana with distinct secondary peaks occurring in regular intervals. These reads also show a nonrandom distribution of di-pyrimidines (the substrate for CPD formation) with TT enrichment at positions 7-8 of the reads. Therefore, UV damage to mtDNA appears to result in production of DNA fragments of characteristic lengths and positions relative to the damaged location. The mechanisms producing these fragments are unclear, but we hypothesize that they result from a previously uncharacterized DNA degradation pathway or repair mechanism in mitochondria.

Estimating bloodstain age in the short term based on DNA fragment length using nanopore sequencer

We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.

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 2290: Integration of cell-free DNA end motifs and fragment lengths can improve identification of active genes in liquid biopsies

Abstract Multiple studies have shown that the plasma cell-free DNA (cfDNA) from cancer patients differs from healthy individuals in both fragment length and fragment end motifs (FEMs). Yet, there is a lack of understanding regarding how the two factors combined can be associated with cancer and gene transcription. In this study, we evaluated cfDNA fragmentomics in plasma from lung cancer patients (n = 17) and healthy individuals (n = 7) using targeted sequencing (lung cancer: n = 12, healthy: n = 7) and whole-genome sequencing (WGS) (lung cancer: n = 5). A personal gene expression profile was established with H3K36me3 cell-free chromatin immunoprecipitation sequencing (cfChIP-seq). cfChIP-seq isolates nucleosomes with histone marks of active transcription directly from plasma, meaning cfDNA enrichment with cfChIP reflects gene activity in the cells of origin. Short cfDNA fragments were isolated using in vitro size-selection. Somatic mutations were identified from input cfDNA through the AVENIO pipeline. The findings were validated in an external public dataset comprising of WGS-cfDNA data from healthy individuals. Cancer patients contain a larger fraction of short (<150bp) cfDNA fragments compared to healthy individuals. Cancer patients also demonstrate an enrichment of distinct FEMs compared to healthy individuals. Investigating the association between gene activity and fragment lengths revealed that genes with high expression display an enrichment of short cfDNA fragments (median = 19.99%, IQR: 16.94% - 27.13%, P < 0.0001) compared to genes with low expression. Furthermore, distinct GC-rich FEMs are enriched in active genes. Combining the frequency of short cfDNA fragments with the presence of distinct FEMs results in a further enrichment of the highest expressed genes (median = 37.85%, IQR: 30.10% - 39.49%, P < 0.0001). In vitro size selection of short cfDNA also results in enrichment of GC-rich FEMs. In vitro size-selection can isolate cfDNA representing active genes and in vitro size-selection enrichment correlates with the cfChIP-seq enrichment (Spearman’s ρ range: 0.499-0.882). This study expands the knowledge regarding cfDNA fragmentomics. We document how cancer patients and healthy individuals have different fragmentomic features. In addition, we shed new light on how gene activity in the cells of origin is associated with cfDNA fragment lengths and FEMs. Together, the results can increase the utility of liquid biopsies by determining gene activity in cancer patients. Citation Format: Christoffer Trier Maansson, Louise Skov Thomsen, Peter Meldgaard, Anders Lade Nielsen, Boe Sandahl Sorensen. Integration of cell-free DNA end motifs and fragment lengths can improve identification of active genes in liquid biopsies [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 2290.

Integration of Cell-Free DNA End Motifs and Fragment Lengths Can Identify Active Genes in Liquid Biopsies.

Multiple studies have shown that cell-free DNA (cfDNA) from cancer patients differ in both fragment length and fragment end motif (FEM) from healthy individuals, yet there is a lack of understanding of how the two factors combined are associated with cancer and gene transcription. In this study, we conducted cfDNA fragmentomics evaluations using plasma from lung cancer patients (n = 12) and healthy individuals (n = 7). A personal gene expression profile was established from plasma using H3K36me3 cell-free chromatin immunoprecipitation sequencing (cfChIP-seq). The genes with the highest expression displayed an enrichment of short cfDNA fragments (median = 19.99%, IQR: 16.94-27.13%, p < 0.0001) compared to the genes with low expression. Furthermore, distinct GC-rich FEMs were enriched after cfChIP. Combining the frequency of short cfDNA fragments with the presence of distinct FEMs resulted in an even further enrichment of the most expressed genes (median = 37.85%, IQR: 30.10-39.49%, p < 0.0001). An in vitro size selection of <150 bp cfDNA could isolate cfDNA representing active genes and the size-selection enrichment correlated with the cfChIP-seq enrichment (Spearman r range: 0.499-0.882, p < 0.0001). This study expands the knowledge regarding cfDNA fragmentomics and sheds new light on how gene activity is associated with both cfDNA fragment lengths and distinct FEMs.

Preserving morphology while extracting DNA: a non-destructive field-to-museum protocol for slide-mounted specimens.

Our study aimed to develop an optimised laboratory protocol ensuring the preservation of morphological structures and extraction of high-quality DNA sequences from Psychodidae (Insecta, Diptera) specimens. With 310 analysed specimens, we investigated the impact of distinct laboratory treatments by employing two shaking categories (constant and interrupted) with five different incubation periods (16, 12, 8, 4 and 2 hours) during the DNA extraction process. Notably, 80.65% of the specimens exhibited morphological changes during DNA extraction. Our results indicated no statistical difference between constant and interrupted shaking for the total of morphological structures lost. However, within each shaking category, the loss of structures was influenced significantly by the incubation period. Prolonged incubation correlated with increased structural losses, whereas shorter incubation periods caused minor alterations in structures lost. In addition, our results showed a significant difference between constant and interrupted shaking treatments for DNA concentration. Likewise, the incubation period showed differences within each shaking category. Successful COI sequencing was achieved in 89.6% of specimens, with negligible differences in DNA fragment lengths across treatments. Our findings underscore the importance of an optimised protocol and its potential in systematic research involving nematoceran dipteran specimens by balancing morphological integrity and DNA extraction efficiency.

Generation of Whole-Genome Bisulfite Sequencing Libraries for Comprehensive DNA Methylome Analysis.

Whole-genome bisulfite sequencing (WGBS) enables the detection of DNA methylation at a single base-pair resolution. The treatment of DNA with sodium bisulfite allows the discrimination of methylated and unmethylated cytosines, but the power of this technology can be limited by the input amounts of DNA and the length of DNA fragments due to DNA damage caused by the desulfonation process. Here, we describe a WGBS library preparation protocol that minimizes the loss and damage of DNA, generating high-quality libraries amplified with fewer polymerase chain reaction (PCR) cycles, and hence data with fewer PCR duplicates, from lower amounts of input material. Briefly, genomic DNA is sheared, end-repaired, 3'-adenylated, and ligated to adaptors with fewer clean-up steps in between, minimizing DNA loss. The adapter-ligated DNA is then treated with sodium bisulfite and amplified with a few PCR cycles to reach the yield needed for sequencing.

Salivary microbial diversity – an investigation on possible biomarkers for polycystic ovarian syndrome from eastern India

Introduction and aim. Polycystic ovarian syndrome (PCOS) is one of the major reproductive health issues, thought to be multifactorial, needs serious attention as a dual burden (health and economic) mainly for developing countries like India, due to its rapid rise (30%) in the last couple of years. Therefore, widespread and liberal screening for this disorder towards prognosis, diagnosis and intervention seems to be an urgent area of research. In this background, the present study attempts to unravel the association of salivary microbial diversity and PCOS. Material and methods. To achieve the purpose 100 clinically diagnosed PCOS individuals and 110 age matched non-PCOS participants from Bengalee Hindu caste population, West Bengal, India was considered. Obtained salivary samples were identified with 16S rDNA amplification and microbial diversity were determined by Alu I restriction enzyme digestion. Results. The present study revealed an explicit pattern of DNA fragment lengths varied between 200 bp and 225 bp in PCOs in comparison to the non-PCOS group. Conclusion. The cardinal feature of the present study as the first attempt from India envisaged, utilization of salivary microbial diversity as an additional potential and economizing biomarker for PCOS that stimulate new horizon of research in 21st century’s anthropology – the anthropology of microbes.

Inactivation of the TIM complex components leads to a decrease in the level of DNA import into Arabidopsis mitochondria.

The phenomenon of DNA import into mitochondria has been shown for all major groups of eukaryotes. In plants and animals, DNA import seems to occur in different ways. It has been known that nucleic acids enter plant organelles through alternative channels, depending on the size of the imported molecules. Mitochondrial import of small DNA (up to 300 bp) partially overlaps with the mechanism of tRNA import, at least at the level of the outer membrane. It is noteworthy that, in plants, tRNA import involves components of the protein import apparatus, whose role in DNA transport has not yet been studied. In this work, we studied the role of individual components of the TIM inner membrane translocase in the process of DNA import into isolated Arabidopsis mitochondria and their possible association with the porin VDAC1. Using knockout mutants for the genes encoding Tim17 or Tim23 protein isoforms, we demonstrated for the first time the involvement of these proteins in the import of DNA fragments of different lengths. In addition, inhibition of transport channels with specific antibodies to VDAC1 led to a decrease in the level of DNA import into wild-type mitochondria, which made it possible to establish the specific involvement of this porin isoform in DNA import. In the tim17-1 knockout mutant, there was an additional decrease in the efficiency of DNA import in the presence of antibodies to VDAC1 compared to the wild type line. The results obtained indicate the involvement of the Tim17-1 and Tim23-2 proteins in the mechanism of DNA import into plant mitochondria. At the same time, Tim23-2 may be part of the channel formed with the participation of VDAC1, while Tim17-1, apparently, is involved in an alternative DNA import pathway independent of VDAC1. The identification of membrane carrier proteins involved in various DNA import pathways will make it possible to use the natural ability of mitochondria to import DNA as a convenient biotechnological tool for transforming the mitochondrial genome.

An ultrasonic biosample disruptor with two triangular teeth on its radiation face.

Ultrasound has been used in biosample disruption such as disruption of algal cell and DNA. New structure of ultrasonic biosample disruptor (UBD) needs to be explored to increase the energy efficiency. In this study, an UBD with two triangular teeth on the bottom radiation face of the water tank has been proposed, to concentrate the acoustic energy into the slot between the two neighboring triangular teeth, in order to raise the acoustic energy utilization and fragmentation performance. The acoustic energy concentration into the slot is verified by the FEM computation, and the improvement of fragmentation performance is experimentally confirmed with spirulina and tribonema, compared to the traditional UBD which has a flat radiation face. The number proportion of fragment in the length range of 10-20μm generated by the UBD proposed in this work is 17.08% and 10.82% more than that generated by the traditional UBD for the two samples, respectively. Besides, the UBD proposed in this work has a much smaller standard deviation of DNA fragment length (47bp) than the traditional UBD (249bp), with a similar mean length of fragments. Moreover, the maximum weight proportion of fragment in the range of 100-300bp, generated by the UBD proposed in this work, is 71.4%.

Unlocking the genomes of formalin‐fixed freshwater fish specimens: An assessment of factors influencing DNA extraction quantity and quality

AbstractObjectiveRecent technological developments may facilitate the description of evolutionary relationships and population genetic structure as well as other information relevant to fisheries management by using readily available natural history collections. Contemporary sequence capture and short‐read sequencing methods offer opportunities to analyze highly fragmented DNA from formalin‐fixed specimens so long as enough DNA of sufficient quality is recovered.MethodsWe compared two protocols developed to extract DNA from formalin‐fixed tissues using specimens of three freshwater fishes: the Southern Brook Lamprey Ichthyomyzon gagei, Slimy Sculpin Cottus cognatus, and Brown Trout Salmo trutta. Extractions were attempted using hot alkali digestion with and without buffer wash pretreatments to compare the DNA concentration, purity, and fragment length of DNA recovered between extraction protocols, tissue types (muscle and caudal fin tissue for Brown Trout and Slimy Sculpin), and preservation periods (5 or 7 years for Southern Brook Lamprey).ResultLikelihood models generally did not detect DNA quantity differences between extraction protocols nor tissue types; however, 6.0–8.7× more DNA was recovered from Slimy Sculpin caudal fins than from muscle tissue. Extraction protocol had mixed effects on DNA purity; the wash protocol outperformed the no‐wash protocol for Slimy Sculpin and Brown Trout, but the reverse was true for the lamprey. Purer DNA was recovered from the caudal fins; however, fragment lengths were generally greater from muscle tissue for both ray‐finned species.ConclusionOur results suggest that the best tissue for sampling may depend on the quality metric considered most important for a study's objectives and that omitting time‐consuming tissue wash steps can yield DNA of quantity and quality comparable to DNA from more complex methods. Regardless of species, the DNA extracted from most samples using both protocols met quantity and quality thresholds that are likely to result in short‐read sequencing success. These results provide optimism for unlocking the wealth of genetic information in natural history collections for use in fisheries management and conservation genomics.

Assessment for Identification of Stelechocarpus burahol and Sister Species Complex of Annonaceae Family Using trnL Intron Sequences

Stelechocarpus burahol (kepel) belongs to the Annonaceae family, and is considered to be a native species to Indonesia which is mainly distributed on the island of Java. However, the plant’s existence is currently difficult to find, so it is categorized as rare in Indonesia. A molecular approach using DNA barcoding technique is significant to assist plant identification. The gene that is widely used and proven to be accurate for Annonaceae is trnL-F. This study was aimed to evaluate the efficiency of trnL as DNA barcode for the identification of S. burahol and its relatives (Annonaceae) from Java Island. In total 10 specimens have been used in this study. Whole genome DNA was isolated by Tiangen kit and amplified by Polymerase Chain Reaction (PCR) technique using a specific primer. Results showed that trnL was easily amplified with a DNA fragment length of 500-600 bp. The trnL amplicons have successfully sequenced as indicated by the high QV20+ values. The sequence compositions were high in AT bases (63.9%). BLAST analysis of the sequences showed that S. burahol and sister species have been confirmed its identity according to the reference sequences in NCBI with query cover identities 98%-100%. This research can be concluded that trnL-F is suitable and recommended as a DNA barcode for S. burahol and its relatives. However, further research is suggested to combine analysis of both coding (rbcL, matK, etc) and non-coding (trnL) markers for better identification results.

ПОРОДНІ ОСОБЛИВОСТІ АЛЕЛЬНОГО ПРОФІЛЮ ГЕНІВ PIT1 ТА GH УКРАЇНСЬКОЇ АБОРИГЕННОЇ ЛЕБЕДИНСЬКОЇ ПОРОДИ КОРІВ

The preservation of genetic diversity is a paramount concern in addressing global challenges today. The foundation of biological diversity hinges on its genetic components, and the loss of species and genetic variability poses a genuine threat to the biosphere. This is because the sustainability of natural ecosystems and agroecosystems depends directly on their genetic adaptability to changing environmental conditions.&#x0D; In cattle, genes associated with milk yield play a crucial role, including genes related to milk proteins and hormones. Among these, the pituitary-specific transcription factor (PIT1) and growth hormone (GH) genes are of particular significance. In this study, we investigated the polymorphism of these genes in the Lebedyn breed of cattle.&#x0D; A total of 2 gene polymorphisms, PIT1 and GH, were examined through molecular and genetic research conducted at the genetics laboratory of the Institute of Animal Breeding and Genetics named after M. V. Zubets, part of the National Academy of Agrarian Science of Ukraine. The study utilized 32 DNA samples extracted from the venous blood of Lebedyn breed cows using the 'DNA Sorb-B' kit from AmpliSens. Genotyping was carried out through polymerase chain reaction (PCR-RFLP) analysis of restriction fragment length polymorphism, employing the “Tertsyk” amplifier for PCR. The resulting DNA restriction analysis products were assessed via electrophoresis in a 2–3% agarose gel containing ethidium bromide, with DNA fragment lengths determined using the GeneRuler TM 50 bp DNA Ladder and Thermo Scientific™ GeneRuler 1 kb Plus DNA Ladder markers.&#x0D; For the PIT1 gene analysis, a 451 bp amplified fragment was treated with the enzyme HinfI, yielding the following allele frequencies: A (0.38) and B (0.62). The heterozygous AB genotype was the most prevalent (0.45), while homozygotes for the A allele constituted 0.15 of the population. In the case of the GH gene fragment (223 bp), hydrolysis was performed using the restriction enzyme AluI. Analysis of allele frequency distribution indicated a high frequency of the L allele (0.85), with no animals exhibiting the homozygous GHVV genotype, and the GHLL genotype was present in 0.7 of the population.&#x0D; These findings reveal unique features of the allelic spectrum of the PIT1 (V-0.62) and GH (L-0.85) genes specific to the Ukrainian indigenous Lebedyn breed of cows. Selectionally significant genotypes include PIT1AA (15%) and GHLL (70%), while heterozygote carriers, which contribute to genetic diversity within the population, account for 45% and 30%, respectively.&#x0D; This study’s results hold particular value due to the alarming decline in the population of local indigenous breeds and the imminent threat to the preservation of agricultural genetic resources.

Predicting tumour content of liquid biopsies from cell-free DNA

BackgroundLiquid biopsy is a minimally-invasive method of sampling bodily fluids, capable of revealing evidence of cancer. The distribution of cell-free DNA (cfDNA) fragment lengths has been shown to differ between healthy subjects and cancer patients, whereby the distributional shift correlates with the sample’s tumour content. These fragmentomic data have not yet been utilised to directly quantify the proportion of tumour-derived cfDNA in a liquid biopsy.ResultsWe used statistical learning to predict tumour content from Fourier and wavelet transforms of cfDNA length distributions in samples from 118 cancer patients. The model was validated on an independent dilution series of patient plasma.ConclusionsThis proof of concept suggests that our fragmentomic methodology could be useful for predicting tumour content in liquid biopsies.

Circulating tumour DNA (ctDNA) burden quantified through low-pass whole genome sequencing as an early pharmacodynamic biomarker of therapeutic response in patients enrolled on phase 1 trials.

23 Background: Early phase trials are conducted to determine the recommended dose and safety of an investigational therapy. Targeted therapies and immunotherapy may not always have dose-dependent toxicities. Therefore, dose finding is increasingly reliant on optimal biological dosing. Pharmacodynamic biomarkers are increasingly important to demonstrate proof of concept. Using ctDNA allows for dynamic and early tumour assessments compared to imaging. We sought to evaluate ctDNA as an early pharmacodynamic biomarker of therapeutic response. Methods: Patients (pts) enrolled on phase 1 trials at the National Cancer Centre Singapore were prospectively consented to collect serial blood samples at defined timepoints (pre-treatment, mid-cycle 1, end of cycle 1, and with subsequent imaging evaluations). ctDNA burden was quantified using low pass whole genome sequencing (lpWGS) with both established (ichorCNA) and novel (cell-free DNA degradation) bioinformatic analyses of ctDNA burden, and targeted panel sequencing. Early kinetics of ctDNA burden prior to imaging evaluation was evaluated as response (reduction in ctDNA burden) versus progression (increase in ctDNA burden). Results: Between May 2021 to Oct 2022, 68 plasma samples (median 3, range 2-5) were collected from 22 pts. Median age was 56 years (range 37-77) and 73% were male. Pts were enrolled on trials of targeted therapy (55%), immunotherapy (40%) or combinations of both (5%). Common tumour types were lung (36%), colorectal (23%) and pancreatic (9%) cancer. Best response on phase 1 trial as per imaging and RECIST v1.1 was partial response (PR) in 9%, stable disease (SD) in 45%, progressive disease (PD) in 27% and not evaluable (NE; taken off trial early due to toxicity) in 18%. Quantification of ctDNA burden using cell-free DNA fragment length pattern demonstrated enhanced limit of detection in detecting baseline ctDNA compared to cell-free DNA copy number profile based ichorCNA method (100% vs 59%). For pts on targeted therapies, early kinetics of ctDNA burden indicated ctDNA response in 9/13 pts, with subsequent imaging showing PR (n=2), SD (n=2), PD (n=2) or NE (n=3). In the remaining 4/13 pts on targeted therapies with early ctDNA progression, subsequent imaging showed SD (n=3) or NE (n=1). For pts on immunotherapy, early ctDNA progression was seen in 8/10 pts, with subsequent imaging showing SD (n=5) and PD (n=3). Patient enrolment and sample analysis is ongoing to further validate these initial findings and updated data will be presented. Conclusions: Early ctDNA evaluation via lpWGS may represent an early pharmacodynamic biomarker of therapeutic response. ctDNA has the potential to provide early proof of concept, particularly for targeted therapies, that may influence subsequent decisions for dose determination.

The Need for Speed and Energy Efficiency in Genome Analysis

The Need for Speed and Energy Efficiency in Genome Analysis

Optimizing the lysis step in CTAB DNA extractions of silica-dried and herbarium leaf tissues.

The use of cetyltrimethylammonium bromide (CTAB) is an effective and inexpensive method of extracting DNA from plants. The CTAB protocol is frequently modified to optimize DNA extractions, but experimental approaches rarely perturb a single variable at a time to systematically infer their effect on DNA quantity and quality. We investigated how chemical additives, incubation temperature, and lysis duration affected DNA quantity and quality. Altering those parameters influenced DNA concentrations and fragment lengths, but only extractant purity was significantly affected. CTAB and CTAB plus polyvinylpyrrolidone buffers produced the highest DNA quality and quantity. Extractions from silica gel-preserved tissues had significantly higher DNA yield, longer DNA fragments, and purer extractants compared to herbarium-preserved tissues. We recommend DNA extractions of silica gel-preserved tissues that include a shorter and cooler lysis step, which results in purer extractions compared to a longer and hotter lysis step, while preventing fragmentation and reducing time.

Surfactant-Free Decellularization of Porcine Auricular Cartilage Using Liquefied Dimethyl Ether and DNase.

The most common decellularization method involves lipid removal using surfactant sodium dodecyl sulfate (SDS) and DNA fragmentation using DNase, and is associated with residual SDS. We previously proposed a decellularization method for the porcine aorta and ostrich carotid artery using liquefied dimethyl ether (DME), which is free from the concerns associated with SDS residues, instead of SDS. In this study, the DME + DNase method was tested on crushed porcine auricular cartilage tissues. Unlike with the porcine aorta and the ostrich carotid artery, it is important to degas the porcine auricular cartilage using an aspirator before DNA fragmentation. Although approximately 90% of the lipids were removed using this method, approximately 2/3 of the water was removed, resulting in a temporary Schiff base reaction. The amount of residual DNA in the tissue was approximately 27 ng/mg dry weight, which is lower than the regulatory value of 50 ng/mg dry weight. Hematoxylin and eosin staining confirmed that cell nuclei were removed from the tissue. Residual DNA fragment length assessment by electrophoresis confirmed that the residual DNA was fragmented to less than 100 bp, which was lower than the regulatory limit of 200 bp. By contrast, in the uncrushed sample, only the surface was decellularized. Thus, although limited to a sample size of approximately 1 mm, liquefied DME can be used to decellularize porcine auricular cartilage. Thus, liquefied DME, with its low persistence and high lipid removal capacity, is an effective alternative to SDS.

Abstract 6700: Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma

Abstract Background: While the incidence of esophageal adenocarcinoma (EAC) is rapidly increasing, the 5-year survival rate of this disease remains poor at less than 25%, with a substantial number of patients exhibiting inherent or acquired resistance to chemotherapy. Circulating tumor DNA (ctDNA) isolated from liquid biopsies of EAC patients holds clinical biomarker potential in this disease, but our understanding of how chemotherapy affects ctDNA release remains limited. Methods: In order to study the impact of cancer treatment on ctDNA emission in vitro, we first sought to establish a chemo-resistant model of EAC. As such, the EAC cell line OE19 was exposed to discontinuous cisplatin treatments for three months in order to establish drug resistance. Following this, established chemo-resistant OE19 cells and parental (chemo-sensitive) OE19 cells were exposed to five daily cisplatin treatments, and cell culture supernatant was collected every day throughout treatment. ctDNA was subsequently isolated from cell culture supernatant and quantified by Qubit fluorometer dsDNA HS assay. ctDNA quantities were additionally assessed by droplet digital PCR (ddPCR) assay using primers and probes targeting the TP53 N310K mutation in the OE19 cell line. Finally, ctDNA fragment lengths were analyzed using the Agilent Bioanalyzer 2100. Results: A chemo-resistant model of the OE19 cell line was established, as exemplified by marked morphological changes in the cells and significantly increased cell viability determined by CCK8 assay after 48-hour treatments of various doses of cisplatin (p&amp;lt;0.05). ctDNA levels, as measured by both Qubit fluorometer and mutant copies detected by ddPCR assay, increased after chemotherapy treatment for both chemo-sensitive and resistant cells. Notably, ctDNA emission was higher with larger amounts cell death, with chemo-sensitive cells emitting significantly higher levels of ctDNA compared to chemo-resistant cells throughout treatment (p&amp;lt;0.05). Fragmentomics analysis revealed ctDNA peaks corresponding to apoptosis (~167 bp) and necrosis (~10,000 bp). Moreover, chemotherapy treatment caused a shift in average fragment size, with larger fragments being observed during chemotherapy treatment in both chemo-sensitive and resistant cells. Conclusion: This study reveals that in vitro cancer models can be used to successfully study ctDNA emission during pre-clinical drug analyses. ctDNA release was found to be correlated to cytotoxicity and cell death, providing us with valuable insight into how clinical liquid biopsy data should be interpreted. Finally, fragment size analysis of ctDNA reveals a potential novel biomarker for treatment response in cancer patients. Citation Format: Alexandra Bartolomucci, Sarah Tadhg Ferrier, Thupten Tsering, Xin Su, Jonathan Cools-Lartigue, Julia V. Burnier. Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6700.