Total Human DNA Research Articles

Assessing DNA Degradation through Differential Amplification Efficiency of Total Human and Human Male DNA in a Forensic qPCR Assay.

The assessment of degradation is crucial for the analysis of human DNA samples isolated from forensic specimens. Forensic quantitative PCR (qPCR) assays can include multiple targets of varying amplicon size that display differential amplification efficiency, and thus different concentrations, in the presence of degradation. The possibility of deriving information on DNA degradation was evaluated in a forensic qPCR assay not specifically designed to detect DNA fragmentation, the Plexor HY (Promega), by calculating the ratio between the estimated concentrations of autosomal (99 bp) and Y-chromosomal (133 bp) targets ("[Auto]/[Y]"). The [Auto]/[Y] ratio measured in 57 formalin-fixed, paraffin-embedded samples was compared to a quality score (QS) calculated for corresponding STR profiles using quantitative data (allele peak height). A statistically significant inverse correlation was observed between [Auto]/[Y] and QS (R = -0.65, p < 0.001). The [Auto]/[Y] values were highly correlated (R = 0.75, p < 0.001) with the "[Auto]/[D]" values obtained using the PowerQuant (Promega) assay, expressly designed to detect DNA degradation through simultaneous quantification of a short (Auto) and a long (D) autosomal target. These results indicate that it is possible to estimate DNA degradation in male samples through Plexor HY data and suggest an alternative strategy for laboratories lacking the equipment required for the assessment of DNA integrity through dedicated qPCR assays.

Quantification of total human DNA using qPCR in mixture samples of Crimes Against Sexual Freedom to estimate the success of analysis with autosomal STRs and Y-STRs

ABSTRACT In this study, 241 mixed autosomal STR genetic profiles (swabs − 172, slides − 69) from processed casework samples were analysed. From these samples, DNA was extracted and quantified by qPCR; the results were analysed with the PowerQuant Analysis Software. The extracted DNA samples were amplified with the Investigator 24Plex QS and VeriFiler kits and separated and detected in an ABI 3500 Genetic Analyzer. A quantification correlation was observed between the total autosomal DNA targets [Auto] and the Y chromosome target [Y] in mixed samples in which the success of amplification of the male autosomal STRs markers was associated with high values of the quantification of the target of [Y] and low DNA ratio values [Auto]/[Y] (p = 0.00 < 0.05). Samples with [Auto]/[Y] ratio values much higher than the estimated value required analysis with Y-STR markers. The study herein, which assessed a relatively large number of sexual assault samples, demonstrates that quantitation ratio values of DNA mixtures effectively contribute to the decision-making process on whether and what genetic marker system should be selected for analysis of a male component in unknown casework samples.

"Total Human DNA Sampling" – Forensic DNA profiles from large areas

Employed for the first time in 1986, DNA profiling is nowadays established as one of the most widely used forensic techniques worldwide. However, until today, no efficient sampling technique existed to collect DNA from human skin cells from a large area, not to say from the floor of an entire room. This has been extremely unfortunate, as there is enormous forensic potential in these DNA traces from the ground to provide clues as to who has been present at a particular location, i.e. at the crime scene. By desquamation, humans loose several millions of skin cells per day, everywhere they stand, sit or walk; and they can do little about it. We developed a fast and simple method by which we can make use of all those lost skin cells. We use a vacuum cleaner equipped with a specialized filter cartridge to sample the ground. Fragmentation of the filter membrane and subsequent parallel processing of the filter fragments, using a modified Chelex® 100 extraction protocol, significantly reduce the complexity of the dust mixture. In this way, a large number of interpretable major contributor DNA profiles can be generated from individuals who have been present on the sampled surface. Overall, at least 38 % of the generated DNA profiles from all sampled test areas fulfilled the criteria for submission of single major contributor profiles to the Swiss DNA database. As demonstrated through a mock crime scene scenario simulating an indoor stabbing event, the perpetrator's DNA could be found on the floor even after a very short stay in the room of less than one minute. Furthermore, already the first application of the method at a real crime scene led to relevant case information for the police. Given its large investigative potential, we recommend Total Human DNA Sampling as a helpful complemental forensic tool to conventional DNA trace collection in major crimes.

Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology.

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the linker sequence for the mature LASSO probe through Cre-LoxP intramolecular recombination. This strategy generates high quality LASSO probes libraries (≈46% of correct probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 Escherichia coli ORFs spanning from 400- to 5000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, were able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

Twenty-Seven Y-Chromosome Short Tandem Repeats Analysis of Italian Mummies of the 16th and 18th Centuries: An Interdisciplinary Research.

Roccapelago (MO) is a small village located in the Northern Central Apennines, with a population of 31 inhabitants (2014). In 2010, more than 400 individuals dated between the end of the 16th and the 18th century, many of which partially mummified, were discovered in the crypt of the church. This small village, because of its geographical location and surrounding environment, seems to possess the characteristics of a genetic isolate, useful for population genetics and genealogical analyses. Thus, a diachronic study of DNA aimed at investigating the structure and dynamics of the population of Roccapelago over the about 4 centuries, was conducted by analyzing ancient and modern inhabitants of the village. The 14 modern samples were selected by considering both the founder surnames of the village, identified thanks to the study of parish registers, and the grandparent’s criterion. From 25 ancient mummies, morphologically assigned to male individuals, the petrous bone, that harbors high DNA amounts, was selected for the DNA extraction. The quantification and qualitative assessment of total human male DNA were evaluated by a real-time PCR assay using the Quantifiler Trio DNA Quantification Kit and multiplex PCR of 27 Y-chromosome short tandem repeat (Y-STR) markers included in the Yfiler Plus PCR Amplification Kit, with seven rapidly mutating Y-STR loci for improving discrimination of male lineages, was performed to genotype the samples. Y-STRs were analyzed according to the criteria of ancient DNA (aDNA) analysis to ensure that authentic DNA typing results were obtained from these ancient samples. The molecular analysis showed the usefulness of the Y chromosome to identify historically relevant remains and discover patterns of relatedness in communities moving from anthropology to genetic genealogy and forensics.

Development and forensic validation of human genomic DNA quantification kit.

DNA quantification is an essential step for successful multiplex short tandem repeat (STR) polymerase chain reactions (PCR), which are used for confirming identities using human genomic DNA. The new DNA quantification kit, named the National Forensic Service Quantification (NFSQ) kit, simultaneously provides total human DNA concentration, human male DNA concentration, and a DNA degradation index (DI) using multiplex TaqMan fluorescent probes. The NFSQ was validated according to developmental validation guidelines from the SWGDAM and MIQE. NFSQ detected up to 0.00128ng/μL and could detect male DNA up to a 1:8000 ratio of male to female DNA. In PCR inhibitor tests, NFSQ could measure DNA at a concentration of 200ng/μL of humic acid and 600μM of hematin. The NFSQ kit showed a DI value trend similar to other qPCR kits. In the reproducibility study, the coefficient of variation of the NFSQ kit was within 10%. The quantitative results of the casework samples obtained using the NFSQ kit were consistent with the STR interpretation results. The NFSQ kit can be useful in the human identification process, as it has detection capabilities similar to those of other comparable quantification kits.

Optimizing DNA recovery and forensic typing of degraded blood and dental remains using a specialized extraction method, comprehensive qPCR sample characterization, and massively parallel sequencing

Human dental remains encountered in criminal casework evidence, missing person cases, or mass disaster tragedies provide a valuable sample source for DNA typing when suitable soft tissue is unavailable. Using traditional methods, teeth samples can be challenging to process, resulting in low-quantity and/or quality nuclear DNA and insufficient profiles for comparisons. This study examines the performance of a three-part nuclear DNA analysis workflow for teeth samples based on (1) improved dental tissue recovery using the Dental Forensic Kit (DFKMR) (Universidad de los Andes) and DNA extraction with QuickExtract™ FFPE DNA Extraction Kit (Lucigen®), (2) quantification with InnoQuant® HY (InnoGenomics Technologies) for sensitive assessment of total human and male DNA quantity/quality, and (3) massively parallel sequencing for simultaneous genotyping of 231 short tandem repeat (STR) and single-nucleotide polymorphism (SNP) markers with the ForenSeq® DNA Signature Prep Kit (Verogen, Inc.). Initial evaluation of artificially degraded blood samples (n = 10) achieved highly sensitive and informative quantification results with InnoQuant® HY, enabling successful first pass genotyping with the MiSeq FGx® System. Twenty-three STR alleles (out of 85) and 70 identity informative SNP loci (out of 94) were recovered from two pg total long target DNA input (0.86 ng total short target input) and an InnoQuant degradation index (DI) of 460 (severely degraded). The three-part workflow was subsequently applied to teeth samples (dental pulp, root cement tissues; n = 13) with postmortem intervals (PMI) of the teeth ranging from 8 days to approximately 6 months. Informative SNP and STR DNA profiles were obtained, to include 78 STR alleles and 85 identity informative SNP loci typed (of 94 total SNP targets) in a 1 month, four-day PMI root cement sample with one pg total long target DNA input and a DI of 76. These data indicate successful performance of the proposed workflow from degraded DNA from teeth samples.

Inferring the presence of spermatozoa in forensic samples based on male DNA fractionation following differential extraction

To address sexual assault kit backlogs some laboratories in North America have implemented ‘Direct to DNA’ (DTD) approaches for the examination of relevant vaginal, oral, rectal and external genitalia swabs from sexual assault examination kits. Using this approach no preliminary serological screening for semen or spermatozoa is performed. Instead, swabs are directly subjected to differential extraction and quantitation using a dual quantification system. Decisions regarding the next steps in processing each sample are typically based on the quantity of male DNA detected, the fraction in which it is detected, and its ratio to the total human DNA in the sample.In the absence of serological results it remains of value in many cases to determine whether spermatozoa are present in the sample and whether the male DNA profile may be attributed to this body fluid. In this study we examine the distribution of male DNA from various body fluids between epithelial and spermatozoa fractions following differential extraction. Based on these results we identified criteria under which a DNA profile can be reliably attributed to spermatozoa.A total of 18 blood samples, 129 saliva samples, and 78 semen samples were processed. The maximum amount of male DNA observed in the sperm fraction of a spermatozoa-free sample was 10.4 ng and the maximum portion of total male DNA observed in the sperm fraction was 7.7%. In contrast, when a sample contained spermatozoa the minimum portion of total male DNA observed in the sperm fraction was 52.6%. This research supports a 50% threshold of male DNA in the sperm fraction following differential extraction as a conservative criterion under which the scientist at the Centre of Forensic Sciences (CFS) may infer that spermatozoa is present in a sample in the absence of serological results. This general threshold is applicable to all sample types (underwear, clothing, swabs, condoms) tested.We further demonstrate that, if the same male DNA profile is represented more prominently in the sperm fraction versus the epithelial fraction, the analyst should not fall into the intuitive trap of inferring the presence of spermatozoa in the sample. Instead, enrichment calculations must be based on the measured quantity of male and total DNA in each fraction and not the male:female DNA ratios observed in the DNA profiles.

PO-349 Identification of differentially expressed lncRNAs in metformin resistant SH-SY5Y neuroblastoma cells

IntroductionOf the total human DNA only 1.2% o codes for proteins, with the rest only being transcribed into regulatory RNA, known as non-coding RNA. Long non-coding RNAs (lncRNAs) are non-coding RNAs that have no or limited protein-coding potential, are >200 nt in length and include- among other categories- long/large intergenic/intervening RNAs (lincRNAs), intronic lncRNAs, natural antisense transcripts (NATs) and pseudogenes. lncRNAs are key regulators of several cellular processes and have been associated with a variety of diseases, including cancer. Metformin (N, N-dimethyl biguanide) is an oral biguanide already in clinical use against diabetes and has also been suggested to decrease the incidence of various cancers, including neuroblastoma, the most common extracranial paediatric cancer arising at the sympathetic nervous system.Material and methodsThe present study aimed at assessing the differential expression of protein-coding and, primarily, non-protein-coding RNAs between control untreated SH-SY5Y neuroblastoma cells and cells resistant to 3 mM Metformin via a paired-end RNA sequencing approach.Results and discussionsIn the first data set (untreated cells compared to cells resistant to 3 mM Metformin), out of 13 741 genes measured, 7855 genes were found to be differentially expressed with 108 of them falling within the lncRNA category. Among these 33 comprise lincRNAs, 24 are NATs and 18 pseudogenes. In the second data set (untreated cells compared to cells treated with 20 mM Metformin) 13 481 genes were tested, of which 5652 showed perturbed expression. Among them, 86 belong to the lncRNA category, and in particular, 33 are lincRNAs, 19 are NATs and 13 are pseudogenes. Interestingly, 34 of the assessed lncRNAs display differential expression in both data sets. The results were obtained using a threshold of 0.05 for statistical significance (p-value) and a log fold change of expression with an absolute value of at least 0.6.Neuroblastoma cells’ response to Metformin, as well as the acquisition of resistance to the drug, trigger the differential expression of a great diversity of lncRNAs.ConclusionGiven that Metformin is an appealing and promising therapeutic approach against neuroblastoma, these lncRNAs could, in turn, be used as molecular biomarkers towards better prediction, prognosis and diagnosis of the disease. Moreover, such an approach would be of interest as part of a combination therapy.

Genome-wide association study for SNPs associated with PCOS in human patients.

This study investigated the possible association between single nucleotide polymorphism (SNP) sites on a genome wide level and the presence of polycystic ovary syndrome (PCOS) in a local population. Patients treated for PCOS in the outpatient clinic of the reproductive medicine center of Changzhou Maternal and Child Health Care Hospital (affiliated to Nanjing Medical University) from January of 2010 to December 2012 were selected. Female patients affected by infertility due to simple oviduct reasons or male factors, during the same period, were enrolled for the control group. A genome-wide association study was performed. Specific experimental steps included extraction of the total human DNA and optimization of PCR amplification of target genes; flight mass spectrometry for genotyping; and statistical analyses of sequencing results. By primary selection and secondary verification at two stages in the experiment, three SNP sites were found to contain significantly different allele frequencies between the patient and control groups (P<0.05): rs346795081 on THADA, rs346803513 on DENND1A and rs346999236 on TOX3. The average expression levels at the three discovered SNPs sites were significantly different between the patient and the control groups, indicating their correlation with PCOS, and the possible role of their corresponding genes on the pathogenesis of the disease.

Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets

The development and validation of InnoQuant® HY, a real-time PCR system containing four DNA targets—two RE autosomal targets of different sizes, male specific targets, and an internal positive control target—are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition.The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow can be created that minimizes unnecessary sample processing and retesting while maximizing recovery of probative DNA profiles from challenging biological evidence.

Development of a quantitation approach for total human and male DNA based on real time PCR followed by high resolution melting analysis.

We developed and validated a total human DNA quantitation technique that simultaneously allows male DNA detection. This assay, called Amel-Y, is a duplex Real Time PCR followed by HRM (high resolution melting) analysis using the intercalating dye SYTO9. Amel-Y duplex produces two amplicons, one for the amelogenin gene (106/112 bp, female/male) and another (84 bp) corresponding to human Y chromosome-specific fragment to detect male DNA. After HRM analysis, two melting peaks differing in 5.3°C-5.5°C are detected if both male and female DNA are present and only one if only female DNA is present. For specificity assessment, the inclusion of high concentrations of bacterial and fungal DNA in the quantitation reactions allowed discarding species cross-reactivity. A set of crime scene evidence from forensic casework has been quantified with commercial kits and compared with Amel-Y duplex. Our method detected male DNA from a concentration of 18 pg/μL and supports autosomal/Y DNA detection ratio up to 200:1. A limitation of the technique is its inability to quantify male and female donnors in a mixed sample. The Amel-Y duplex demonstrated to be an efficient system for quantifying total human DNA being a specific, rapid, sensitive, and cost-effective method suitable for mixed DNA samples and applicable to any field where human DNA quantification is required, such as molecular diagnosis, population genetics, and forensic identification.

Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant¨r) system

Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant¨r) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant¨r) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay tm)s specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.

The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology

Recent culture-independent studies have enabled detailed mapping of human microbiome that has not been hitherto achievable by culture-based methods. DNA extraction is a key element of bacterial culture-independent studies that critically impacts on the outcome of the detected microbial profile. Despite the variations in DNA extraction methods described in the literature, no standardized technique is available for the purpose of microbiome profiling. Hence, standardization of DNA extraction methods is urgently needed to yield comparable data from different studies. We examined the effect of eight different cell lysis protocols on the yield and quality of the extracted DNA from oral rinse samples. These samples were exposed to cell lysis techniques based on enzymatic, mechanical, and a combination of enzymatic–mechanical methods. The outcome measures evaluated were total bacterial population, Firmicutes levels and human DNA contamination (in terms of surrogate GAPDH levels). We noted that all three parameters were significantly affected by the method of cell lysis employed. Although the highest yield of gDNA was obtained using lysozyme-achromopeptidase method, the lysozyme-zirconium beads method yielded the peak quantity of total bacterial DNA and Firmicutes with a lower degree of GAPDH contamination compared with the other methods. Taken together our data clearly points to an urgent need for a consensus, standardized DNA extraction technique to evaluate the oral microbiome using oral rinse samples. Further, if Firmicutes levels are the focus of investigation in oral rinse microbiome analyses then the lysozyme-zirconium bead method would be the method of choice in preference to others.

Developmental validation of the Quantifiler® HP and Trio Kits for human DNA quantification in forensic samples

The quantification of human genomic DNA is a necessary first step in the DNA casework sample analysis workflow. DNA quantification determines optimal sample input amounts for subsequent STR (short tandem repeat) genotyping procedures, as well as being a useful screening tool to identify samples most likely to provide probative genotypic evidence. To better mesh with the capabilities of newest-generation STR analysis assays, the Quantifiler® HP and Quantifiler® Trio DNA Quantification Kits were designed for greater detection sensitivity and more robust performance with samples that contain PCR inhibitors or degraded DNA. The new DNA quantification kits use multiplex TaqMan® assay-based fluorescent probe technology to simultaneously quantify up to three human genomic targets, allowing samples to be assessed for total human DNA, male contributor (i.e., Y-chromosome) DNA, as well as a determination of DNA degradation state. The Quantifiler HP and Trio Kits use multiple-copy loci to allow for significantly improved sensitivity compared to earlier-generation kits that employ single-copy target loci. The kits’ improved performance provides better predictive ability for results with downstream, newest-generation STR assays, and their shortened time-to-result allows more efficient integration into the forensic casework analysis workflow.

Menstrual cycle phase at the time of rape does not affect recovery of semen or amplification of STR profiles of a suspect in vaginal swabs

The effect of women menstrual cycle on the forensic analysis of rapes was studied in a random group of 170 victims aged among 10 and 51 years. Participants were grouped according to the day of the menstrual cycle in which they were at the moment of the assault. From each participant, samples of vaginal fluid were taken and analyzed for sperm cells, p30 protein, total human DNA and human male DNA. Moreover, amplification of suspect's autosomal STR and Y-STR was attempted. Suspects’ autosomal STR profiles were obtained from 92 of the 101 samples in which spermatozoa were found; and Y-STR haplotype was obtained in 1 of the 9 samples where autosomal STR profiles of a male were not obtained. On the other hand, Y-STR haplotypes were obtained in 2 of the 21 samples negative for sperm cells but positive for p30 protein. Y-STR haplotypes were also obtained in 11 of the 48 samples negative for sperm cells and p30 protein. It was found that groups of participants did not differ on the recovery of sperm cells from the vaginal swabs, quantification of suspect's DNA or amplification of their STR profiles. It is concluded that the menstrual cycle phase at the moment of the sexual assault does not affect the main outcomes of the forensic investigation of rapes.

Validation of automated PCR-setup of the Quantifiler® Trio DNA Quantification kit on the Biomek® 4000 Laboratory Automation Workstation

The accurate quantification of DNA is critical for choosing the optimal workflow for evidence samples in forensic genetics. Thermo Fisher Scientific has recently released the Quantifiler® Trio DNA Quantification kit, which they announce has several improvements compared to previous quantification kits, such as increased accuracy in determining the level of total human and male DNA in low-template samples. Since automation of PCR-setups reduces the manual workload and the risk for human errors, we have chosen to validate the PCR-setup of the Quantifiler® Trio kit on a small benchtop liquid handler, the Biomek® 4000 (Beckman Coulter). Particularly, we tested the reproducibility of the kit and its performance on low-template samples, focusing on the human and male targets. Furthermore, we conducted a mixture study with different male to female ratios, correlating quantification results to DNA-profiles (NGM Select™) of these mixtures. In respect to automation, we have also examined the level of cross contamination during plate setup. Our results show that the Biomek® 4000 is as suited as the manual procedure for setup of the Quantifiler® Trio. No evidence of cross contamination was observed. We found that the automated setup of the Quantifler® Trio kit on the Biomek® 4000 results in consistent and reliable quantification results, both for total human DNA and male DNA. Furthermore, the Biomek® 4000 represents a flexible and user-friendly platform for automated PCR-setups. Implementation of the Quantifiler® Trio seems to improve the STR-typing workflow for low-template samples and mixtures with a minor male contributor in focus of investigation.

Quantifiler® Trio DNA validation and usefulness in casework samples

DNA profiling with sets of highly polymorphic STR markers is applied in various aspects of human identification. During the past few years significant progress has been made in solving technical challenges associated with STR profiling, including the ability to analyze degraded DNA, low amounts of DNA and/or the presence of PCR inhibitors. Quantifiler® Trio DNA method enables forensic laboratories to simultaneously obtain a quantitative and qualitative assessment of total human and human male DNA. Moreover, this kit has a set of primers to amplify and detected two autosomal multiple-copy target loci, known as the small autosomal (SA) and large autosomal (LA), which provide a quality index (DI) to detect the presence of degraded DNA along with PCR inhibitors. The experiments focus on kit performance parameters relevant to the intended use of the kits as DNA quantification assays and also application in casework studies. The workflow combining the DNA quantification with Quantifiler® Trio DNA quantification kit and STR amplification has the potencial to improve forensic laboratories efficiency.

Abstract PL02-01: Stool DNA detection of colorectal neoplasia: A new high bar for noninvasive screening

Abstract PL02-01: Stool DNA detection of colorectal neoplasia: A new high bar for noninvasive screening

Analysis of circulating DNA and protein biomarkers to predict the clinical activity of regorafenib and assess prognosis in patients with metastatic colorectal cancer: a retrospective, exploratory analysis of the CORRECT trial

Tumour mutational status is an important determinant of the response of metastatic colorectal cancer to targeted treatments. However, the genotype of the tissue obtained at the time of diagnosis might not accurately represent tumour genotype after multiple lines of treatment. This retrospective exploratory analysis investigated the clinical activity of regorafenib in biomarker subgroups of the CORRECT study population defined by tumour mutational status or plasma protein levels. We used BEAMing technology to identify KRAS, PIK3CA, and BRAF mutations in DNA obtained from the plasma of 503 patients with metastatic colorectal cancer who enrolled in the CORRECT trial. We quantified total human genomic DNA isolated from plasma samples for 503 patients using a modified version of human long interspersed nuclear element-1 (LINE-1) quantitive real-time PCR. We also measured the concentration of 15 proteins of interest-angiopoietin 2, interleukin 6, interleukin 8, placental growth factor, soluble TIE-1, soluble VEGFR1, VEGF-A, VEGF-C, VEGF-D, VEGF-A isoform 121, bone morphogenetic protein 7, macrophage colony-stimulating factor, stromal cell-derived factor-1, tissue inhibitor of metalloproteinase 2, and von Willebrand factor-in plasma samples from 611 patients. We did correlative analyses of overall survival and progression-free survival in patient subgroups based on mutational status, circulating DNA concentration, and protein concentrations. The CORRECT trial was registered with ClinicalTrials.gov, number NCT01103323. Tumour-associated mutations were readily detected with BEAMing of plasma DNA, with KRAS mutations identified in 349 (69%) of 503 patients, PIK3CA mutations in 84 (17%) of 503 patients, and BRAF mutations in 17 (3%) of 502 patients. We did not do correlative analysis based on BRAF genotype because of the low mutational frequency detected for this gene. Some of the most prevalent individual hot-spot mutations we identified included: KRAS (KRAS G12D, 116 [28%] of 413 mutations; G12V, 72 [17%]; and G13D, 67 [16%]) and PIK3CA (PIK3CA E542K, 27 [30%] of 89 mutations; E545K, 37 [42%]; and H1047R, 12 [14%]). 41 (48%) of 86 patients who had received anti-EGFR therapy and whose archival tumour tissue DNA was KRAS wild-type in BEAMing analysis were identified as having KRAS mutations in BEAMing analysis of fresh plasma DNA. Correlative analyses suggest a clinical benefit favouring regorafenib across patient subgroups defined by KRAS and PIK3CA mutational status (progression-free survival with regorafenib vs placebo: hazard ratio [HR] 0·52, 95% CI 0·35-0·76 for KRAS wild-type; HR 0·51, 95% CI 0·40-0·65 for KRAS mutant [KRAS wild type vs mutant, pinteraction=0·74]; HR 0·50, 95% CI 0·40-0·63 for PIK3CA wild-type; HR 0·54, 95% CI 0·32-0·89 for PIK3CA mutant [PIK3CA wild-type vs mutant, pinteraction=0·85]) or circulating DNA concentration (progression-free survival with regorafenib vs placebo: HR 0·53, 95% CI 0·40-0·71, for low circulating DNA concentrations; HR 0·52, 95% CI 0·40-0·70, for high circulating DNA concentrations; low vs high circulating DNA, pinteraction=0·601). With the exception of von Willebrand factor, assessed with the median cutoff method, plasma protein concentrations were also not associated with regorafenib activity in terms of progression-free survival. In univariable analyses, the only plasma protein that was associated with overall survival was TIE-1, high concentrations of which were associated with longer overall survival compared with low TIE-1 concentrations. This association was not significant in multivariable analyses. BEAMing of circulating DNA could be a viable approach for non-invasive analysis of tumour genotype in real time and for the identification of potentially clinically relevant mutations that are not detected in archival tissue. Additionally, the results show that regorafenib seems to be consistently associated with a clinical benefit in a range of patient subgroups based on mutational status and protein biomarker concentrations. Bayer HealthCare Pharmaceuticals.