Detection Of Rare Mutations Research Articles

Detecting ultra low-frequency variants and gene fusions in lung cancer patients using an amplicon-based Firefly NGS method.

e23062 Background: The analysis of EGFR, KRAS, and BRAF mutations and Alk fusion is critical for guiding targeted therapy selection, detecting drug resistance, and monitoring residual disease in patients with NSCLC. Designing next-generation sequencing (NGS) assays for detecting low-frequency variants, however, is an ongoing challenge. The limited availability of cfDNA combined with the breadth of coverage necessary to create meaningful, clinically-actionable results requires a solution with multiplex capacity which, in turn, requires greater technological sensitivity and specificity. Here we aim to develop such a solution: an ultra-accurate NGS technology using concatmer-based error correction with amplicon workflow for fast detection of rare mutations including SNV and fusion. Methods: We developed an amplicon-based panel covering variants of EGFR, BRAF, and KRAS, as well as a panel to detect Alk fusion. CfDNA simulate and cfDNA from healthy individuals were used to test assay sensitivity and specificity. Further validation was performed via a comparative analysis of 64 late-stage lung cancer patients using both Firefly -Comet and ddPCR. Results: Analytical sensitivity of the EGFR-TKI 3-gene panel was 100% detection at an allele frequency of 0.1% for 20ng of cfDNA input. Similarly, analytical sensitivity of the Alk fusion panel was 75% detection at an allele frequency of 0.1% and 100% at an allele frequency of 0.25% for the same input. Among our patient cohort, 5 EGFR variants (19del, T790M, L858R, G719X, L861X) and 2 KRAS variant (G12X) were detected. Firefly-Comet demonstrated strong per-variant detection-rate concordance ( > 99%) compared to ddPCR results. The PPV is 100% and the NPV is 98.7%. Statistical analysis of reported allele frequency concordance between Firefly-Comet and ddPCR reveals R-Sq > 0.9. Conclusions: In summary, we have developed Firefly-Comet, an easy-to-use amplicon-based NGS assay capable of detecting single-digit copies of somatic mutants and gene fusions in cfDNA. The multiplex capacity of Firefly-Comet makes it well-suited for supporting targeted therapy selection, drug resistance detection, and treatment monitoring.

Current and Emerging Applications of Droplet Digital PCR in Oncology.

The clinical management of cancer has evolved in recent years towards more personalized strategies that require a comprehensive knowledge of the complex molecular features involved in tumor growth and evolution, and the development of drug resistance mechanisms leading to disease progression. Droplet digital PCR (ddPCR) has become one of the most accurate and reliable tools for the examination of genetic alterations in a wide variety of cancers because of its high sensitivity and specificity. ddPCR is currently being applied for absolute allele quantification, rare mutation detection, analysis of copy number variations, DNA methylation, and gene rearrangements in different kinds of clinical samples. This methodology has proven useful for the evaluation of archival tumor tissues, where poor DNA quality and limited sample availability are major obstacles for standard methods, providing less subjective and more automated quantitative results. However, most applications of ddPCR in cancer are focused on liquid biopsies (including cell-free DNA as well as circulating tumor cells) because these represent non-invasive alternatives to tissue biopsies that can more accurately reflect intratumoral heterogeneity and track the dynamic changes in tumor burden that occur in response to treatment at different times during follow-up. A broad spectrum of molecular markers have been interrogated in blood using ddPCR for diagnostic, predictive, and monitoring purposes in various malignancies. Emerging alternative approaches using other body fluids such as cerebrospinal fluid and urine are also currently being developed. This article aims to give a complete overview of ddPCR applications for molecular screening in oncology.

Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.

Single, long molecule PCR for the detection of rare mutations in Mitochondrial DNA

IntroductionOocytes contain the whole mitochondrial genomes of mammals, and the inheritance pattern of mitochondrial DNA in oocytes do not follow Mendel's rules of heredity. Mitochondrial DNA is maternally inherited in the oocytes, and only mother's mitochondrial genome can be passed to the next generation. The mitochondrial genome is a closed, circular piece of DNA which has a length of about 16kb, and there are hundreds to hundreds of thousands mitochondrial DNA in a single cell. Mitochondrial DNA mutation rate is generally difficult to detect and is often over‐estimated. This research is focusing on developing a sequencing strategy to accurately get mitochondrial DNA mutation rate of single, long molecules from a wild type mouse germline.Research ObjectivesThe research goal is to develop an efficient and accurate way to assess the mutation rate of mitochondrial DNA from wild type mouse oocytes.MethodsWe isolated and individually lysed single oocytes from a wild type female mouse. A serial dilution of the DNA sample was made to find its working concentration for generating single, long molecules, and prepared a PCR reaction by using the specific DNA sample concentration. A high‐fidelity DNA polymerase was used to generate 16kb PCR products originating from single template molecules. Sanger sequencing was used to sequence these DNA molecules.ResultsWe used single molecule PCR to amplify the mitochondrial DNA from three wild type mouse oocytes and sequenced all the PCR products, yielding a total coverage about 330,392bp. Twenty‐two mitochondrial genomes from three oocytes were sequenced, with an average length of about 15,017bp. Mouse mitochondrial DNA length is 16,299kb, so each of our mitochondrial DNA molecule has an average coverage about 92.13% of the whole mitochondrial DNA genome. Only two mutations were found in two separate mitochondrial genomes, and the resulting mutation rate of mitochondrial DNA in the oocytes was 6.05×10^‐6. This is a very low mutation rate across over 300,000bp.ConclusionThe mutation rate of mitochondrial DNA in wild type mouse germline is remarkable low in our research by using single, long molecule PCR. This PCR method allows for the accurate detection of mutations in the mouse mitochondrial DNA in oocytes. Our data indicates that, by using single, long molecule PCR combined with Sanger sequencing, we can accurately get informations from a molecule that has the most coverage of its genome. Additionally, we used a high‐fidelity enzyme to serve as DNA polymerase in the PCR reaction, thus preventing the chance of artificial mutations from the beginning of the PCR reaction. With the right procedure of our sequencing strategy, the rare mutations and the mutation rate in single germline cells can be accurately determined.Support or Funding InformationEllison Medical Foundation; NIH R37‐AG012279

From single-molecule detection to next-generation sequencing: microfluidic droplets for high-throughput nucleic acid analysis.

Droplet-based microfluidic technologies have proved themselves to be of significant utility in the performance of high-throughput chemical and biological experiments. By encapsulating and isolating reagents within femtoliter–nanoliter droplet, millions of (bio) chemical reactions can be processed in a parallel fashion and on ultra-short timescales. Recent applications of such technologies to genetic analysis have suggested significant utility in low-cost, efficient and rapid workflows for DNA amplification, rare mutation detection, antibody screening and next-generation sequencing. To this end, we describe and highlight some of the most interesting recent developments and applications of droplet-based microfluidics in the broad area of nucleic acid analysis. In addition, we also present a cursory description of some of the most essential functional components, which allow the creation of integrated and complex workflows based on flowing streams of droplets.

Wild-type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS

Mutant KRAS proto-oncogene GTPase (KRAS) serves an important role in predicting the development, diagnosis, treatment and efficacy of targeted drug therapies for colorectal cancer. To improve the detection efficacy of trace amount of mutant KRAS, the locked nucleic acid-based method was modified in the present study. Internal competitive amplification fragments were used to improve the inhibition of wild-type KRAS with a wild-type blocking (WTB) probe and specifically amplify the trace amounts of mutant KRAS. The modified method, quantitative clamp-based polymerase chain reaction technology using WTB coupled with internal competitive reference to enhance the amplification specificity, named WIRE-PCR, completely blocked the amplification of wild-type KRAS in 50–150 ng DNA templates. The added internal competitive amplified fragments were amplified together with the target gene, which were used to reduce base mismatch due to the high number of cycles in PCR and quantify the total amount of DNA. The results demonstrated that WIRE-PCR facilitated the detection of mutated alleles at a single molecular level. In the colorectal biopsies from 50 patients with suspected colorectal cancer, 18 cases (36%) contained mutant KRAS, and the amount of mutant DNA accounted for 18.6–64.2% of the total DNA. WIRE-PCR is a simple, rapid and low-cost quantitative analysis method for the detection of trace amounts of the mutant KRAS.

A microfluidic chip based on surfactant-doped polydimethylsiloxane (PDMS) in a sandwich configuration for low-cost and robust digital PCR

Digital PCR (dPCR) is a powerful method for the absolute quantification and rare mutation detection of nucleic acids. However, the available dPCR platforms require either expensive instruments or complicated operation, which limit their extensive application. This paper describes a robust self-partition chip to perform dPCR in a very simple and inexpensive format. The chip is constructed by sandwiching a micromolded surfactant-doped PDMS layer between two glass slides, containing 10,000 reaction compartments (0.785nL each). The two simple strategies, “glass-PDMS-glass” sandwich configuration and surfactant-doped surface modification, are used to enhance PCR compatibility and enable efficient amplification in the chip by minimizing water loss and nonspecific adsorption of protein. Furthermore, powered by pre-degassing of its PDMS substrate and an attached PDMS slab, the chip autonomously loads and partitions sample into a high-density array of microwells for performing dPCR analysis. We evaluated the quantitative capabilities of the dPCR chip by measuring a ten-fold serial dilution of genomic DNA from lung cancer cell line H1975. Finally, we also demonstrate its feasibility in the accurate measurement of small copy number variations, which exhibited better accuracy than real-time quantitative PCR (qPCR). Such simple and low-cost platform promises to accelerate the widespread use of dPCR.

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations.

Detection of Rare Mutations by Routine Analysis of KRAS, NRAS, and BRAF Oncogenes.

Molecular genetic analysis of KRAS, NRAS, and BRAF genes was carried out in order to develop an optimal algorithm for detection of minor mutations. We analyzed 35 melanoma and 33 colorectal cancer specimens. Frequent G12D/V/A/C/S mutations were detected in KRAS. The most frequent BRAF mutation in melanoma was V600E, the percentage of rare mutations is significant for DNA diagnosis (24%). Identification of rare BRAF mutations 1790C→G (L597R), 1798_1799delinsAA (V600K), 1798_1799delinsAG (V600R), and 1799_1800delinsAA (V600E) and NRAS mutation 38G→T (G13V) was possible only by Sanger sequencing. The combination of real-time PCR and sequencing can improve analysis sensitivity and ensure concordance of the tested loci with the international recommendations.

Beta-Binomial Model for the Detection of Rare Mutations in Pooled Next-Generation Sequencing Experiments.

Against diminishing costs, next-generation sequencing (NGS) still remains expensive for studies with a large number of individuals. As cost saving, sequencing genome of pools containing multiple samples might be used. Currently, there are many software available for the detection of single-nucleotide polymorphisms (SNPs). Sensitivity and specificity depend on the model used and data analyzed, indicating that all software have space for improvement. We use beta-binomial model to detect rare mutations in untagged pooled NGS experiments. We propose a multireference framework for pooled data with ability being specific up to two patients affected by neuromuscular disorders (NMD). We assessed the results comparing with The Genome Analysis Toolkit (GATK), CRISP, SNVer, and FreeBayes. Our results show that the multireference approach applying beta-binomial model is accurate in predicting rare mutations at 0.01 fraction. Finally, we explored the concordance of mutations between the model and software, checking their involvement in any NMD-related gene. We detected seven novel SNPs, for which the functional analysis produced enriched terms related to locomotion and musculature.

Microparticle-based RT-qPCR for highly selective rare mutation detection

The quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become one of the most widely used methods in the detection of disease-specific RNAs. The RT-qPCR involves two separate steps, RT and qPCR. In this study, we suggest a new RT-qPCR protocol with the particles of primer-immobilized networks (PINs), performing capture, RT and amplification of a target RNA in one particle. The production of undesired cDNAs was dramatically suppressed by the specific capture of the target RNA within the particle. Afterward, RT and amplification processes are performed without loss of cDNAs as exchanging the reaction solution. The biomarker gene of chronic myeloid leukemia, Bcr-Abl fusion transcript, is detected in the sensitivity of single mutant leukemic cell mixed in 104 normal cell using this protocol with the excellent restraint of non-specific signal. This protocol that whole processes are performed in the particle in a row is preferred for the highly specific detection of target RNAs in complex sample.

Abstract 403: Performance assessment of highly sensitive NGS assay for TP53

Abstract We have designed a highly sensitive assay based on the Safe-Sequencing technology(1) to detect de novo mutations in the TP53 gene. A custom panel was designed to cover 95% of all reported mutations in TP53. To demonstrate assay performance, we assessed LoD, LoB, reproducibility, and repeatability and evaluated concordance with BEAMing(2). A customized data analysis pipeline for ultra-deep sequencing runs was developed that includes extensive quality control and advanced mutation calling. This highly sensitive NGS-based workflow can be applied to monitor recurrent or minimal residual disease in cancer patients after surgery or chemotherapy.

Abstract 402: Multiplex TaqMan assays for rare mutation analysis using digital PCR

Abstract Introduction Detection of rare mutations for research purposes in tumor tissue and cell free DNA (cfDNA) allows for monitoring of tumor progression and regression. cfDNA isolated from plasma combined with a sensitive detection method like digital PCR is non-invasive and enables earlier detection compared to conventional imaging techniques. Building on the TaqMan based Rare Mutation assay set for detection of rare mutations using digital PCR on the QuantStudio 3D Digital PCR System, we are now developing multiplex assays for simultaneous detection of several mutations. We selected relevant mutations in the EGFR and KRAS genes for our initial multiplex application: EGFR G719, EGFR exon 19 deletions, and KRAS G12/G13. These mutations may have implications for potential future targeted therapy. Methods Primers and probes of singleplex Rare Mutation Assays were reformulated to generate multiplex assays detecting the EGFR and KRAS mutations. All multiplex assays were tested on template composed of wild-type genomic DNA background mixed with mutant plasmid reflecting each of the mutations detected by the multiplex assays. Summary Initial experimental results were successful and showed excellent signal intensity and clear cluster separation when analyzed with the QuantStudio 3D AnalysisSuite™ Cloud Software. The EGFR G719 mutations (COSM6239, COSM6253, COSM6252) were detected using a 3plex assay, EGFR exon 19 deletions (COSM12383, COSM12422, COSM12678, COSM6223, COSM6254, COSM6255) were detected using a 6plex assay, and KRAS G12/G13 mutations (COSM516, COSM517, COSM518, COSM520, COSM521, COSM522, COSM527, COSM532) were detected using an 8plex. Conclusion Multiplexing assays for three relevant mutation loci proved feasible and presents an efficient way to assess the presence and the percentage of mutations at these loci. Citation Format: Marion -. Laig, Frances Chan, Le Lac, Ted Straub, Kamini Varma, David Keys. Multiplex TaqMan assays for rare mutation analysis using digital PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 402.

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

Measurements of Intrahost Viral Diversity Are Extremely Sensitive to Systematic Errors in Variant Calling

With next-generation sequencing technologies, it is now feasible to efficiently sequence patient-derived virus populations at a depth of coverage sufficient to detect rare variants. However, each sequencing platform has characteristic error profiles, and sample collection, target amplification, and library preparation are additional processes whereby errors are introduced and propagated. Many studies account for these errors by using ad hoc quality thresholds and/or previously published statistical algorithms. Despite common usage, the majority of these approaches have not been validated under conditions that characterize many studies of intrahost diversity. Here, we use defined populations of influenza virus to mimic the diversity and titer typically found in patient-derived samples. We identified single-nucleotide variants using two commonly employed variant callers, DeepSNV and LoFreq. We found that the accuracy of these variant callers was lower than expected and exquisitely sensitive to the input titer. Small reductions in specificity had a significant impact on the number of minority variants identified and subsequent measures of diversity. We were able to increase the specificity of DeepSNV to >99.95% by applying an empirically validated set of quality thresholds. When applied to a set of influenza virus samples from a household-based cohort study, these changes resulted in a 10-fold reduction in measurements of viral diversity. We have made our sequence data and analysis code available so that others may improve on our work and use our data set to benchmark their own bioinformatics pipelines. Our work demonstrates that inadequate quality control and validation can lead to significant overestimation of intrahost diversity. Advances in sequencing technology have made it feasible to sequence patient-derived viral samples at a level sufficient for detection of rare mutations. These high-throughput, cost-effective methods are revolutionizing the study of within-host viral diversity. However, the techniques are error prone, and the methods commonly used to control for these errors have not been validated under the conditions that characterize patient-derived samples. Here, we show that these conditions affect measurements of viral diversity. We found that the accuracy of previously benchmarked analysis pipelines was greatly reduced under patient-derived conditions. By carefully validating our sequencing analysis using known control samples, we were able to identify biases in our method and to improve our accuracy to acceptable levels. Application of our modified pipeline to a set of influenza virus samples from a cohort study provided a realistic picture of intrahost diversity and suggested the need for rigorous quality control in such studies.

Detection of a rare mutation in the ferroportin gene through targeted next generation sequencing.

Hereditary haemochromatosis (HH) is a disorder characterised by increase of serum iron parameters and gradual iron accumulation in parenchymal organs. Since the discovery of the genetic defects of the most common form of hereditary haemochromatosis (type 1 haemochromatosis [HFE]), many observations have shown that not only rare/familial mutations in HFE can be present but also that mutations in other genes (transferrin receptor 2 [TFR2], hepcidin [HAMP], hemojuvelin [HJV], and ferroportin [SLC40A1]) can lead to rarer genetic forms of iron overload, referred as non-HFE haemochromatosis1. The genetic heterogeneity is particularly evident in the Italian population where only two-thirds of haemochromatosis patients are HFE C282Y homozygotes2, requiring expensive and time-consuming gene specific genotyping to define the molecular diagnosis. Therefore, “second level” genetic tests should be developed for a rapid and simultaneous study of the haemochromatosis genes. The most common non-HFE haemochromatosis is the autosomal dominant haemochromatosis type 4 (HH4), caused by mutations in the SLC40A1 gene, encoding for cellular iron exporter ferroportin. Ferroportin acts as receptor of hepcidin, the key regulator of iron metabolism. The hepcidin/ferroportin interaction induces the internalisation of the complex, causing a decrease in iron export and its retention in the cell3. HH4 can be phenotypically classified in two groups: patients may have hyperferritinaemia with normal/low transferrin saturation (type-A HH4, ferroportin disease) or both serum iron parameters increased (type-B HH4, non-classical ferroportin disease)4. This depends on ferroportin impairment that could be classified as “loss-of-function” or “gain-of-function”. In the first case (type-A), the mutated protein is not expressed on the membrane and it is not able to exert its exporting function. In the second case (type-B), the mutated iron exporter becomes resistant to the activity of hepcidin, causing increased iron absorption. Several mutations have been characterised in HH4 patients, spreading along the entire gene sequence4. The position of the correspondent amino acidic change is important to define the HH4 clinical phenotype5. Among the different ferroportin mutations, the p.A69T variant, despite the causal nucleotide change not being annotated in dbSNP, has been described in a 52-year old Italian woman with diabetes and type-B HH4, on the basis of hepatocyte iron overload6, but no iron parameters or clinical history of the patient were described. The pathogenic role of this mutation has been subsequently confirmed through in vitro experiments in which mutant cDNA has been over-expressed7. These studies demonstrated that p.A69T mutated ferroportin has a partial resistance to hepcidin. Here we report an Italian patient with a severe iron overload phenotype in whom a careful clinical characterisation led to a diagnose of p.A69T non-classical ferroportin disease through the combination of capture of technology with a next generation sequencing (NGS) platform.

A web-oriented software for the optimization of pooled experiments in NGS for detection of rare mutations.

BackgroundThe cost per patient of next generation sequencing for detection of rare mutations may be significantly reduced using pooled experiments. Recently, some techniques have been proposed for the planning of pooled experiments and for the optimal allocation of patients into pools. However, the lack of a user friendly resource for planning the design of pooled experiments forces the scientists to do frequent, complex and long computations.ResultsOPENDoRM is a powerful collection of novel mathematical algorithms usable via an intuitive graphical user interface. It enables researchers to speed up the planning of their routine experiments, as well as, to support scientists without specific bioinformatics expertises. Users can automatically carry out analysis in terms of costs associated with the optimal allocation of patients in pools. They are also able to choose between three distinct pooling mathematical methods, each of which also suggests the optimal configuration for the submitted experiment. Importantly, in order to keep track of the performed experiments, users can save and export the results of their experiments in standard tabular and charts contents.ConclusionOPENDoRM is a freely available web-oriented application for the planning of pooled NGS experiments, available at: http://www-labgtp.na.icar.cnr.it/OPENDoRM. Its easy and intuitive graphical user interface enables researchers to plan theirs experiments using novel algorithms, and to interactively visualize the results.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-1889-6) contains supplementary material, which is available to authorized users.

Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR

Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, preamplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications.

Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing

Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective preventive screening procedure to detect adenomatous polyps, the precursors to CRC, is colonoscopy. Since every colorectal cancer starts as a polyp, detecting all polyps and removing them is crucial. By exactly doing that, colonoscopy reduces CRC incidence by 80%, however it is an invasive procedure that might have unpleasant and, in rare occasions, dangerous side effects. Despite numerous efforts over the past two decades, a non-invasive screening method for the general population with detection rates for adenomas and CRC similar to that of colonoscopy has not yet been established. Recent advances in next generation sequencing technologies have yet to be successfully applied to this problem, because the detection of rare mutations has been hindered by the systematic biases due to sequencing context and the base calling quality of NGS.We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS) to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC.

Next-Generation Genotyping by Digital PCR to Detect and Quantify the BRAF V600E Mutation in Melanoma Biopsies

The detection of the BRAF V600E mutation in melanoma samples is used to select patients who should respond to BRAF inhibitors. Different techniques are routinely used to determine BRAF status in clinical samples. However, low tumor cellularity and tumor heterogeneity can affect the sensitivity of somatic mutation detection. Digital PCR (dPCR) is a next-generation genotyping method that clonally amplifies nucleic acids and allows the detection and quantification of rare mutations. Our aim was to evaluate the clinical routine performance of a new dPCR-based test to detect and quantify BRAF mutation load in 47 paraffin-embedded cutaneous melanoma biopsies. We compared the results obtained by dPCR with high-resolution melting curve analysis and pyrosequencing or with one of the allele-specific PCR methods available on the market. dPCR showed the lowest limit of detection. dPCR and allele-specific amplification detected the highest number of mutated samples. For the BRAF mutation load quantification both dPCR and pyrosequencing gave similar results with strong disparities in allele frequencies in the 47 tumor samples under study (from 0.7% to 79% of BRAF V600E mutations/sample). In conclusion, the four methods showed a high degree of concordance. dPCR was the more-sensitive method to reliably and easily detect mutations. Both pyrosequencing and dPCR could quantify the mutation load in heterogeneous tumor samples.