Low Amounts Of DNA Research Articles

A Comparative Study of Methyl-BEAMing and Droplet Digital PCR for MGMT Gene Promoter Hypermethylation Detection

Background: O-6-methylguanine-DNA methyltransferase is responsible for the direct repair of O6-methylguanine lesions induced by alkylating agents, including temozolomide. O-6-methylguanine-DNA methyltransferase promoter hypermethylation is a well-established biomarker for temozolomide response in glioblastoma patients, also correlated with therapeutic response in colorectal cancer. Objectives: The ARETHUSA clinical trial aims to stratify colorectal cancer patients based on their mismatch repair status. Mismatch repair-deficient patients are eligible for treatment with immune checkpoint inhibitors (anti-PDL-1), whereas mismatch repair-proficient samples are screened for O-6-methylguanine-DNA methyltransferase promoter methylation to identify those suitable for temozolomide treatment. Methods: In this context, a subset of ARETHUSA metastatic colorectal cancer samples was used to compare two different techniques for assessing O-6-methylguanine-DNA methyltransferase hypermethylation: Methyl-BEAMing, a highly sensitive digital PCR approach that combines emulsion PCR and flow cytometry, and droplet digital PCR, a more automated procedure that enables the rapid, operator-independent analysis of a large number of samples. Results: Our study clearly demonstrates that the results obtained using Methyl-BEAMing and droplet digital PCR are comparable, with both techniques showing similar accuracy, sensitivity, and reproducibility. Conclusions: Digital droplet PCR proved to be an efficient method for detecting gene promoter methylation. However, the Methyl-BEAMing method has proved more sensitive for detecting low quantities of DNA.

Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq) to Study DNA Methylation Using Low Amounts of DNA

Background/Objectives: DNA methylation is a key epigenetic mark involved in regulating gene expression. Aberrant DNA methylation contributes to various human diseases, including cancer, autoimmune disorders, atherosclerosis, and cardiovascular diseases. While whole-genome bisulfite sequencing and methylated DNA immunoprecipitation (MeDIP) are standard techniques for studying DNA methylation, they are typically limited to a few samples per run, making them expensive and low-throughput. Therefore, an automation-friendly method is needed to increase throughput and reduce costs without compromising data quality. Methods and Results: We developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed a 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis. Conclusions: Mx-MeDIP-Seq is a cost-effective and efficient method for high-throughput DNA methylation analysis, offering faster processing and reduced sample requirements. This technique makes DNA methylome analysis more accessible for large-scale studies.

Transcriptome amplification and fusion gene detection using ultra-trace cells derived from cytology specimens

Tumours are complex and despite scientific breakthroughs, there is much to be elucidated about cancer and its mechanisms. By personalising treatment, outcomes can be improved and genetic analysis is key to this as it allows genetic alterations to be identified and treatment to be precisely targeted. Dr Kenji Amemiya is a cytotechnologist and Chief Clinical Laboratory Technician at the Yamanashi Prefectural Central Hospital Genome Analysis Center (GAC). He is working to better understand the complex inner landscape of cancer and therefore improve cancer detection, diagnosis and treatment. Formalin‐fixed paraffin‐embedded (FFPE) specimens are typically used for genetic testing but there are drawbacks to this, including the FFPE DNA becoming damaged during the fixation process with formalin, resulting in low quality and quantity of DNA. Amemiya is collaborating closely with Chairman of the GAC, Professor Emeritus Masao Omata. They are interested in the use of cytology samples instead of tissue samples in genome analysis, as a less invasive method. Survival rates are increased when genomic analysis is utilised in non-small cell lung cancer (NSCLC). In one project, Amemiya, Omata and the team explored the potential of performing NGS analysis with archived cytological specimens (ACSs). They utilised an integrated NGS platform called the Oncomine Dx Target Test Multi-CDx system to compare the quantity and quality of extracted nucleic acids, the sequencing quality control (QC) and the detected variants between ACSs and FFPE tissues. They found that the RNA integrity was higher in ACSs and there was no difference in sequencing results, compared with FFPE tissues.

Internal validation of the Precision ID GlobalFiler NGS STR panel v2 kit with locus-specific analytical threshold, and with special regard to mixtures and low template DNA detection

We performed an internal laboratory validation of the Precision ID GlobalFiler NGS STR panel v2 kit to assist the introduction of the technology into the routine forensic casework practice. The study was designed and evaluated based not only on the key validation standards like sensitivity, stability, reproducibility, repeatability, mixture, and concordance, but we also tested the effect of reduced input DNA, we measured and applied locus-specific analytical threshold values, tested two different PCR cycle conditions, sequence artifacts and stutters were also analysed. During the study we also tested the new method on real casework samples. The sensitivity study confirmed that adding 500 pg template DNA for library preparation can be optimal at base PCR cycle number (that was 24), because the measured average heterozygote balance was not lower than 0.82, and each allele was detected above the analytical threshold. However, contrary to previous communications, increasing the PCR cycle numbers up to 28 has not resulted the significant elevation of the heterozygote imbalance. According to our results, raised PCR cycle condition (i.e. 28) is appropriate at or below 150 pg total input DNA. For most loci, the calculated AT was lower than the manufacturer’s recommended. Applying the newly established ATs with raised PCR cycle conditions the allele detection sensitivity and reliability increased. We observed allele dropouts only at the 15 pg template DNA experiments with 5 % frequency, that is better to previously published studies. This result indicates that this low amount of DNA (i.e. 15 pg) could be a minimum limit of template input for a potentially successful analysis. In the mixture study the minor contributor could be detected up to 1:19 mixture ratio. We detected minor alleles in all measurements and concentrations above the threshold if the template DNA were fixed and only SNP differences were observed between the same alleles of the contributors. To test concordance between the new method and traditional STR genotyping we analysed 58 Hungarian individual samples in parallel. Nearby the detected 248 different length-based alleles on the 31 loci in the sample pool we revealed additional 75 sequence variant alleles, that represent an approximately 23 % increase in the total number of observed alleles. The casework study confirmed that the Precision ID GlobalFiler NGS STR panel v2 kit is effective even in genotyping degraded samples with extremely low levels of DNA, if we apply elevated cycle number for library preparation and use locus-specific analytical thresholds.

Development of two step reverse transcription droplet digital PCR (RT-ddPCR) for simultaneous identification of saliva and semen

Determination of the type of body fluids is essential for crime scene reconstruction and for improving the reliability of expert testimonies. Messenger RNA (mRNA) analysis by reverse transcription quantitative PCR (RT-qPCR) has been used in forensic genetics, particularly for body fluid identification. It is a relative quantification method that compares the Ct values of target and reference gene. Thus, the method is unsuitable for determining exact copy numbers of the target gene. To address this limitation, this study performed body fluid-specific mRNA analysis using two-step reverse transcription droplet digital PCR (RT-ddPCR), which is capable of absolute quantification. We found that RT-ddPCR was accurate and sensitive enough to detect as little as 1.5 copies/μl of complementary DNA (cDNA), making it suitable for application using casework samples. It was also highly specific for body fluids, as non-specific amplification did not occur. In addition, saliva-semen mixtures with ratios ranging from 1:50 to 50:1 were successfully identified. When comparing the results of RT-qPCR and RT-ddPCR, some samples were difficult to interpret because of the high Ct values of RT-qPCR. However, when the same samples were analyzed using RT-ddPCR, saliva and semen were distinctly identified. Thus, RT-ddPCR is useful for mixed samples (e.g., in sexual assault cases) with low amounts of DNA, which often leads to ambiguous results when using RT-qPCR. Other body fluids (e.g., vaginal fluid and menstrual blood) can also be identified by including additional markers. This study demonstrates the potential of RT-ddPCR for applications in forensic science.

Transfer and recovery of DNA and metal particles: A proof-of-concept application of a parallel strategy by DNA and environmental scanning electron microscopy analysis

According to the principle of Locard “Every contact leaves a trace", when touching a surface, a bi-directional transfer of self and non-self-DNA residing on the hands and touched objects can occur. Metals are commonly encountered in forensic evidence and, during hand contact with these surfaces, a transfer of metal particles could occur together with the transfer of human DNA. This study proposes a proof-concept approach for the original detection of metal particles and touch DNA to track the activity performed by a donor and particularly to assess the metallic substrate touched before the contact with a subsequent surface. To this scope, a scenario of contact events was simulated by three volunteers, who participated in fingerprint deposition firstly on copper and then on plastic and glass surfaces. Twenty-four stubs were collected on the hands of volunteers and the secondary surfaces and then analyzed by environmental scanning electron microscopy (ESEM). DNA was quantified only from copper and plastic surfaces. Ten additional volunteers followed the same protocol of deposition on copper and then on plastic surfaces to evaluate DNA transfer only. On 20 touch DNA samples, the copper surface yielded significantly lower DNA amounts, ranging from 0.001 to 0.129 ng/μl, compared to the secondary touched plastic surface, ranging from 0.007 to 0.362 ng/μl. ESEM-EDS analysis showed that copper particles could be abundantly detected on the hands of the volunteers after contact with the copper surface. Particles containing silicates with copper were shown on plastic, while they were only found in 1/3 of samples on glass. Our proof-of-concept study has shown that ESEM-EDS analysis has the potential to detect copper particles transferred to the hands of volunteers during contact with a copper metallic surface and deposited on secondarily touched items. The results suggest that this original ESEM-DNA parallel approach could potentially allow the tracking of DNA transfer and metal particles at a crime scene, although this represents only a first step and further research on a wider casuistry could help to address the interpretation of results given activity level propositions.

Sensitivity and specificity of Nanopore sequencing for detecting carbapenem and 3rd-generation cephalosporin-resistant Enterobacteriaceae in urine samples: Real-time simulation with public antimicrobial resistance gene database

ObjectivesTo evaluate the accuracy of beta-lactamase gene detection directly from urine samples by Nanopore sequencing. MethodsDNA was extracted from bacterial pellets in spun urine. The purified DNA was then sequenced in native form by a Nanopore sequencer (MinION) to identify the organisms and beta-lactamase genes. Results were compared to routine urine cultures and standard antimicrobial susceptibility tests (AST). ResultsWe processed 60 urine samples of which routine cultures grew Enterobacteriaceae, including 28 carbapenem-resistant (CRE), 17 extended-spectrum beta-lactamase (ESBL) or AmpC producing, and 15 non-ESBL/AmpC phenotypes. We excluded 7 samples with extremely low DNA amounts (<1 ng/μl) for a final case of 53 in total. The sensitivity of antimicrobial resistance gene detection within 6 h, the optimal duration from real-time simulation, of Nanopore sequencing for the diagnosis of carbapenem-resistant and ceftriaxone-resistant phenotypes was 73.9 % (95%CI 56.0–91.9 %) and 81.1 % (95%CI 68.5–93.7 %), while the specificity was 96.7 % (95%CI 90.2–100.0 %) and 56.3 % (95%CI 31.9–80.6 %), respectively. The median times for MinION to generate DNA reads containing carbapenemase and ESBL/AmpC genes were 93 min (IQR 17–245.5) and 99 min (IQR 31.25–269.75) after sequencing commencement, respectively. ConclusionsNanopore sequencing can identify bacterial genotypic resistance in urine and may enable clinicians to adjust antimicrobial therapy earlier than routine AST.

FecalSeq enrichment with RAD Sequencing from non-invasive environmental samples holds promise for genetic monitoring of an imperiled lagomorph

Despite advances in genomic sequencing and bioinformatics, conservation genomics is still often hindered by a reliance on non-invasive samples. The presence of exogenous DNA and the low quantity and poor quality of DNA in non-invasive samples have been a roadblock to sequencing, thereby limiting the potential for genomic monitoring of endangered species. Recent molecular advances, such as host DNA enrichment, hold promise for facilitating sequencing from non-invasive samples. We used the FecalSeq method to enrich DNA extracted from wild-collected fecal pellets of the imperiled New England cottontail and identified SNPs from 3RAD Sequencing. We obtained SNPs from rabbit pellets, including pellets that were collected in poor environmental conditions and samples that performed poorly with microsatellites. Measures of sequencing success improved with greater amounts of starting DNA and 32% of samples generated SNP genotypes that passed quality control filtering. Genotyping error rates were high, however, and the approach was unable to consistently distinguish unique individuals or matching genotypes, while it was suitable for recovering the expected population structure. Pairing FecalSeq enrichment with RADseq is a promising low-cost method for monitoring wild populations using non-invasive samples in an environmental context, but it may be better suited for informing conservation through population genomics.

P-530 Precise quantification of DNA in spent culture media sheds light on associations between DNA quantity, embryonic ploidy status and morphological quality

Abstract Study question Is it possible to accurately measure cell-free DNA (cfDNA) quantity in embryo spent culture media, and are amounts correlated with embryo morphological and/or chromosomal status? Summary answer A sensitive DNA quantification method was created, demonstrating that cfDNA quantity is not associated with embryo ploidy, but increases when embryos are of poor morphology. What is known already Preimplantation genetic testing involves embryo biopsy, necessitating specialist equipment and highly trained staff, increasing costs. Additionally, there have been concerns over potential embryo damage to the embryo resulting from biopsy. The discovery of cfDNA in medium in which embryos were cultured, led to suggestions that non-invasive PGT (niPGT) might be possible. However, the amount of cfDNA in media samples, and potential correlations with other aspects of embryo biology, require clarification. We aimed to provide the first accurate measurement of the amount of DNA in spent culture medium, providing insights into whether niPGT is likely to provide acceptable accuracy rates. Study design, size, duration This prospective analysis involved media samples from 151 fresh embryos, which were produced at a single IVF clinic over the course of one year. Embryos were cultured individually, with a medium change on day-3 of development. Trophectoderm biopsy was undertaken on day-5/-6, followed by PGT-A. Embryo morphology was assessed according to ASEBIR criteria (ranked A-C). Participants/materials, setting, methods The culture and biopsy protocols used were standard for the clinic. Media was collected prior to biopsy and underwent a novel quantitative whole genome amplification method, allowing precise measurement of DNA with respect to a standard curve generated using samples containing known amounts of DNA. PGT-A involved the use of a highly validated method, based upon next generation sequencing. Correlation between DNA quantity and embryo morphological quality was also studied. Data were analyzed using ANOVA. Main results and the role of chance There was no difference in the quantity of DNA in media samples associated with euploid and aneuploid embryos (mean DNA quantity and standard deviations were 1.72 ± 3.23 pg and 1.54 ± 3.66 pg for aneuploid and euploid embryos, respectively; p = 0.75). Overall, 91% of media samples contained less than the equivalent of one cell (&amp;lt;5pg of DNA) and almost two-thirds of samples (65%) contained less than 1pg of DNA. Given such low quantities of embryonic DNA, achieving accurate genetic testing is likely to be challenging. Drops that held embryos until day-6 had significantly higher levels of DNA than those associated with culture to day-5 (P &amp;lt; 0.0001), suggesting cfDNA accumulates with time. Thus, longer culture periods are likely to yield more DNA for analysis. Comparing the amount of cfDNA with morphological status revealed a significant association. Embryos of fair quality (C) tended to have higher quantities of DNA in spent culture medium, whereas top-quality embryos (classified as A or B) typically had less (mean and standard deviation of DNA quantity: 1.23 ± 3.14 pg for class A and B versus 2.60 ± 3.85 pg for class C; p &amp;lt; 0.05). This may reflect increased cell death and DNA release in poorer quality embryos. Limitations, reasons for caution This project is limited by its single-centre nature and relatively small sample size. A large-scale, multicentre study would be necessary to confirm these results. Wider implications of the findings It has been hypothesised that cfDNA quantity in culture medium may be predictive of embryo chromosomal status. We found no evidence to support this. Although DNA quantities were higher after culture to day-6, levels remained low, calling into question the ability of niPGT to deliver high accuracy diagnosis of embryos. Trial registration number not applicable

METB-05. CSF LIQUID BIOPSY: PAVING THE WAY FOR MINIMALLY INVASIVE BRAIN TUMOR SURVEILLANCE USING LOW-PASS WHOLE GENOME SEQUENCING

Abstract OBJECTIVE We describe a clinical liquid biopsy platform for disease detection and surveillance in pediatric patients with primary central nervous system (CNS) tumors using low-pass whole genome sequencing (LP-WGS) on cell-free DNA (cfDNA) isolated from cerebrospinal fluid (CSF). METHODS CSF samples were collected during routine clinical procedures. cfDNA was extracted and LP-WGS performed using a validated Illumina NextSeq sequencing with a 37 bp paired-end read configuration. Sequencing results were compared to tumor molecular profiling performed by UW-Oncoplex™, a targeted DNA-based next-generation sequencing panel. RESULTS Sixty-one CSF specimens were collected from 29 individual patients with primary brain tumors across 7 institutions, with samples obtained at initial diagnosis or staging (n=17), during treatment (n=29), at therapy completion (n=4), or at disease recurrence (n=11). Patients with medulloblastoma (n=13), ATRT (n=3), high-grade glioma (n=3), low-grade glioma (n=6), ependymoma (n=1), and high-grade neuroepithelial tumor/ embryonal tumors (n=3) were included. Copy number variants consistent with tumor were detected in 24 samples from a median CSF volume of 2.5 mL (range: 1-8 mL) and 54.6 pg/ul of cfDNA (median, range: 1.3-5,220). 31/61 samples yielded enough DNA for LP-WGS while 30 were insufficient due to absence of tumor DNA (true negatives) or low DNA quantity (technical insufficiency). Among the 10 diagnostic samples collected from patients with MRI evidence of disease at initial staging, LP-WGS was positive in 8/10 (80%), compared to 2/10 (20%) positive by cytologic analysis for malignant cells. All 4 staging samples from patients with leptomeningeal metastasis were positive by LP-WGS. At recurrence, LP-WGS was positive for tumor in 6/8 (75%) of MRI-positive patients. CONCLUSIONS LP-WGS of CSF-derived cfDNA can identify clinically relevant somatic copy number variants, offering a minimally invasive liquid biopsy option for ongoing surveillance in pediatric CNS tumors. Further prospective analysis is needed to mitigate interpretation error and refine clinical applications.

Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

Development and validation of a GT-seq panel for genetic monitoring in a threatened species using minimally invasive sampling.

Minimally invasive samples are often the best option for collecting genetic material from species of conservation concern, but they perform poorly in many genomic sequencing methods due to their tendency to yield low DNA quality and quantity. Genotyping-in-thousands by sequencing (GT-seq) is a powerful amplicon sequencing method that can genotype large numbers of variable-quality samples at a standardized set of single nucleotide polymorphism (SNP) loci. Here, we develop, optimize, and validate a GT-seq panel for the federally threatened northern Idaho ground squirrel (Urocitellus brunneus) to provide a standardized approach for future genetic monitoring and assessment of recovery goals using minimally invasive samples. The optimized panel consists of 224 neutral and 81 putatively adaptive SNPs. DNA collected from buccal swabs from 2016 to 2020 had 73% genotyping success, while samples collected from hair from 2002 to 2006 had little to no DNA remaining and did not genotype successfully. We evaluated our GT-seq panel by measuring genotype discordance rates compared to RADseq and whole-genome sequencing. GT-seq and other sequencing methods had similar population diversity and F ST estimates, but GT-seq consistently called more heterozygotes than expected, resulting in negative F IS values at the population level. Genetic ancestry assignment was consistent when estimated with different sequencing methods and numbers of loci. Our GT-seq panel is an effective and efficient genotyping tool that will aid in the monitoring and recovery of this threatened species, and our results provide insights for applying GT-seq for minimally invasive DNA sampling techniques in other rare animals.

Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method

Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1–15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures.

R-tPA Resistance Is Specific for Platelet-Rich Stroke Thrombi and Can Be Overcome by Targeting Nonfibrin Components.

Resistance to r-tPA (recombinant tissue-type plasminogen activator) is a well-known but poorly understood phenomenon that hampers successful recanalization in patients with acute ischemic stroke. Using clinically relevant thrombi from patients with acute ischemic stroke, we investigated if and how thrombus composition impacts r-tPA-mediated lysis. In addition, we explored strategies to overcome r-tPA resistance. Thrombi were split into 2 parts, 1 of which was used for thrombolysis and the other for detailed histological analysis. Thrombolysis was performed in normal human plasma using r-tPA alone, using r-tPA in combination with DNase-1 or using r-tPA in combination with N,N'-diacetyl-l-cystine. Thrombus lysis was calculated as the percentage of residual thrombus weight compared with its initial weight and the degree of lysis was linked to thrombus composition determined via histology. Interestingly, we found that the efficacy of r-tPA-mediated thrombolysis was strongly correlated with the composition of the thrombi. Thrombi containing high amounts of red blood cells and low amounts of DNA and von Willebrand Factor were efficiently degraded by r-tPA, whereas thrombi containing low amounts of red blood cells and higher amounts of DNA and von Willebrand Factor were resistant to r-tPA. Importantly, combination of r-tPA with DNase-1 or N,N'-diacetyl-l-cystine significantly and specifically improved the lysis of these r-tPA-resistant thrombi. Using patient thrombus material, our results for the first time show that the composition of stroke thrombi largely determines their susceptibility to r-tPA-mediated thrombolysis. Red blood cell-poor thrombi have a specific resistance to r-tPA, which can be overcome by targeting nonfibrin components using DNase-1 or N,N'-diacetyl-l-cystine.

Abstract 2299: More information from limited DNA: simultaneous measurement of genetics, 5hmC and 5mC in cell-free DNA

Abstract Liquid biopsy for profiling of cell free DNA (cfDNA) in blood holds huge promise to transform how we experience and manage cancer by early detection and identification of residual disease and subtype. While early work in liquid biopsy focused on the identification of actionable somatic variations at specific loci, the past decade has seen an expansion into non-genetic features, notably methylation. 5-methylcytosine (5-mC) profiles of cancer are differential from non-cancer at many more loci and so provide a stronger signal. Moreover, recent research has suggested that 5-hydroxymethylcytosine (5-hmC) profiles in cfDNA can be a marker for early cancer. However, a standard blood draw yields an average of only 10ng of cfDNA, presenting the dilemma of how to use limited sample to obtain maximum information. Existing methods for measuring methylation using next-generation sequencing cannot distinguish between 5-mC and 5-hmC and are limited in their ability to detect mutations, with approaches that measure both 5-mC and 5-hmC requiring two separate workflows. Hence, to measure complete genetics, 5-mC and 5-hmC using existing technologies requires three separate workflows, each of which require separate DNA input. We will present a technology which sequences at single base resolution the complete genetic sequence of input DNA fragments integrated with the modification status (unmodified, 5-mC or 5-hmC) for each CpG from low nanogram input quantities of DNA. A hairpin construct is used to create a copy of the original DNA strand. Enzymatic conversion followed by next-generation sequencing enables coupled decoding of bases across the original and copy strand, uniquely reporting one of A, C, G, T, 5-mC, or 5-hmC for each position in the input DNA fragment. Using this technology, we generated whole genome 6-letter data (measuring A, C, G, T, 5-mC, and 5-hmC at single-base resolution across the genome) on cell-free DNA extracted from plasma of healthy volunteers and patients with colorectal cancer at various stages of progression. We demonstrate how the technology can be used to compare 5-mC and 5-hmC methylomic profiles, genomics, and fragmentomics, across different stages of colorectal cancer. In particular, we show how disease progression can be marked by changes in 5-mC and 5-hmC across several loci. We propose that the ability to measure 5-mC and 5-hmC at high accuracy and single base resolution, alongside genomic and fragmentomic profiles, from a limited quantity of DNA will enable greater insight into the ctDNA in plasma and help advance the field of liquid biopsy towards fulfilling its promise. Citation Format: Tom J. Charlesworth, Fabio Puddu, Robert Crawford, Annelie Johansson, Ermira Lleshi, Aurelie Modat, Jamie Scotcher, Michael Wilson, Nicholas Harding, Jean Teyssandier, Jens Fullgrabe, Walraj Gosal, Paidi Creed. More information from limited DNA: simultaneous measurement of genetics, 5hmC and 5mC in cell-free DNA [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 2299.

Abstract 3677: Hybrid capture based sequencing from urinary cell free DNA from colorectal cancer patients

Abstract Background: The study of body fluids other than blood is gaining increasing attention in the field of liquid biopsy. Since urine offers a truly non-invasive sampling method, it is a particularly interesting specimen. Evidence suggests that urine cell-free DNA (ucfDNA) harbors information about renal and bladder cancer. However, not much is known about ucfDNA in colorectal cancer (CRC) patients. Therefore, we aimed to test the feasibility of a hybrid capture based NGS approach with and without preanalytical stabilization of urine samples. Methods: Urine samples were collected from 20 patients with metastatic CRC (mCRC), aliquoted and stored with or without a stabilizing agents (PAXgene Urine Liquid Biopsy Set and Streck urine Preserve) at room temperature. Urine cfDNA was isolated at the day of donation and after 3 days. Matched blood samples were additionally collected in PAXgene Blood ccfDNA Tubes (PreAnalytiX). cfDNA from blood and urine was isolated using the QIAsymphony platform (QIAGEN). cfDNA samples were analyzed using the AVENIO ctDNA Analysis Kit (Roche), a hybrid-capture based approach enriching for 17 clinically relevant genes. Results: While all stabilized ucfDNA yielded high quality libraries, library preparation failed in 66.7% of unstabilized urine samples, demonstrating that without stabilization ucfDNA rapidly degrades after urine donation. In native samples, in which sequencing data could be obtained, sequencing depth was significantly decreased compared to stabilized samples. For stabilized samples, sequence analysis revealed full concordance between urine and plasma for putative germline variants. However, when using less than 50ng of input material of ucfDNA, a low signal-to-noise ratio with a high number of false positive low level variants was observed. We therefore adjusted the limit of detection for ucfDNA to 0.5%, at which CRC-related mutations were detected in ucfDNA in one patient. Although these variants, were not observed in plasma cfDNA, they could be detected in all stabilized urine samples, which hints to true variants. Conclusion: Our data demonstrate that a hybrid-capture based analysis approach is feasible for ucfDNA form CRC patients and that urine may provide complementary information about a patient's tumor that may be missed in plasma. Yet, due to degradation and lower concentrations, immediate stabilization of urine after donation is required. Moreover, the limit of detection needs to be adjusted if low amounts of input DNA is available only. Citation Format: Anna Eberhard, Tina Moser, Leandra Ziegler, Georgios Vlachos, Isaac Lazzeri, Martina Loibner, Armin Gerger, Ellen Heitzer. Hybrid capture based sequencing from urinary cell free DNA from colorectal cancer patients [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 3677.

Simulation-based benchmarking of ancient haplotype inference for detecting population structure

Paleogenomic data has informed us about the movements, growth, and relationships of ancient populations. It has also given us context for medically relevant adaptations that appear in present-day humans due to introgression from other hominids, and it continues to help us characterize the evolutionary history of humans. However, ancient DNA (aDNA) presents several practical challenges as various factors such as deamination, high fragmentation, environmental contamination of aDNA, and low amounts of recoverable endogenous DNA, make aDNA recovery and analysis more difficult than modern DNA. Most studies with aDNA leverage only SNP data, and only a few studies have made inferences on human demographic history based on haplotype data, possibly because haplotype estimation (or phasing) has not yet been systematically evaluated in the context of aDNA. Here, we evaluate how the unique challenges of aDNA can impact phasing and imputation quality, we also present an aDNA simulation pipeline that integrates multiple existing tools, allowing users to specify features of simulated aDNA and the evolutionary history of the simulated populations. We measured phasing error as a function of aDNA quality and demographic history, and found that low phasing error is achievable even for very ancient individuals (∼ 400 generations in the past) as long as contamination and average coverage are adequate. Our results show that population splits or bottleneck events occurring between the reference and phased populations affect phasing quality, with bottlenecks resultingin the highest average error rates. Finally, we found that using estimated haplotypes, even if not completely accurate, is superior to using the simulated genotype data when reconstructing changes in population structure after population splits between present-day and ancient populations. We also find that the imputation of ancient data before phasing can lead to better phasing quality, even in cases where the reference individuals used for imputation are not representative of the ancient individuals.

Simulation-based benchmarking of ancient haplotype inference for detecting population structure

Paleogenomic data has informed us about the movements, growth, and relationships of ancient populations. It has also given us context for medically relevant adaptations that appear in present-day humans due to introgression from other hominids, and it continues to help us characterize the evolutionary history of humans. However, ancient DNA (aDNA) presents several practical challenges as various factors such as deamination, high fragmentation, environmental contamination of aDNA, and low amounts of recoverable endogenous DNA, make aDNA recovery and analysis more difficult than modern DNA. Most studies with aDNA leverage only SNP data, and only a few studies have made inferences on human demographic history based on haplotype data, possibly because haplotype estimation (or phasing) has not yet been systematically evaluated in the context of aDNA. Here, we evaluate how the unique challenges of aDNA can impact phasing and imputation quality, we also present an aDNA simulation pipeline that integrates multiple existing tools, allowing users to specify features of simulated aDNA and the evolutionary history of the simulated populations. We measured phasing error as a function of aDNA quality and demographic history, and found that low phasing error is achievable even for very ancient individuals (∼ 400 generations in the past) as long as contamination and average coverage are adequate. Our results show that population splits or bottleneck events occurring between the reference and phased populations affect phasing quality, with bottlenecks resulting in the highest average error rates. Finally, we found that using estimated haplotypes, even if not completely accurate, is superior to using the simulated genotype data when reconstructing changes in population structure after population splits between present-day and ancient populations. We also find that the imputation of ancient data before phasing can lead to better phasing quality, even in cases where the reference individuals used for imputation are not representative of the ancient individuals.

Optimal filter materials for protist quantification via droplet digital PCR

The use of droplet digital polymerase chain reaction (ddPCR) has greatly improved the quantification of harmful protists, outperforming traditional methods like quantitative PCR. Notably, ddPCR provides enhanced consistency and reproducibility at it resists PCR inhibitors commonly found in environmental DNA samples. This study aimed to determine the most effective filter material for ddPCR protocols by assessing the reproducibility of species-specific gene copy numbers and filtration time across six filter types: cellulose acetate (CA), mixed cellulose ester (MCE), nylon (NY), polycarbonate (PC), polyethersulfone (PES), and polyvinylidene fluoride (PVDF). The study used two species of Chattonella marina complexes as a case study. Filtration rates were slower for NY, PC, and PVDF filters. Moreover, MCE, NY, PES, and PVDF yielded lower DNA amounts than other filters. Importantly, the CA filter exhibited the lowest variance (38–39%) and the highest determination coefficients (R2 = 0.92–0.96), indicating superior performance. These findings suggest that the CA filter is the most suitable for ddPCR quantification of marine protists, offering quick filtration and reliable reproducibility.

Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots.

DNA methylation is an epigenetic modification that plays a key role in several cellular processes mediating the fine regulation of gene expression. Aberrant DNA methylation is observed in a wide range of pathologies, including cancer. Since these DNA modifications are transferred to the cell progenies and are stable over the time, the analysis of DNA methylation status has been proposed for diagnostic and prognostic purposes in cancer. Currently, DNA bisulfite conversion is the gold standard method for the high‑throughput analysis of DNA methylation alterations. However, bisulfite treatment induces DNA fragmentation affecting its quality for the downstream analyses. In this field, it is mandatory to identify novel methods to overcome the limits of conventional approaches. In the present study, the Methylation‑Sensitive Restriction Enzyme‑droplet digital PCR (MSRE‑ddPCR) assay was developed as a novel sensitive method for the analysis of DNA methylation of short genomic regions, combining the MSRE assay with the high‑sensitivity ddPCR and using an exogenous methylation sequence as control. Setup and validation experiments were performed analyzing a methylation hotspot of the Solute Carrier Family 22 Member 17 in DNA samples derived from melanoma cell lines as well as from tissues and serum samples obtained from patients with melanoma and healthy controls. Compared with the standard MSRE approaches, the MSRE‑ddPCR assay is more appropriate for the analysis of DNA methylation (methDNA) in samples with low amounts of DNA (up to 0.651 ng) showing a greater sensitivity. These findings suggested the potential clinical application of MSRE‑ddPCR paving the way to the analysis of other methDNA hotspots in different tumors.