Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR.

Despite the interest in characterizing genomic variation, the presence of large repeats at the breakpoints hinders the analysis of many structural variants. This is especially problematic for inversions, since there is typically no gain or loss of DNA. Here, we tested novel linkage-based droplet digital PCR (ddPCR) assays to study 20 inversions ranging from 3.

RAS/RAF mutations in tumor samples and cell-free DNA from plasma and bone marrow aspirates in multiple myeloma patients.

: To evaluate the detection of gene mutations in bone marrow biopsy and circulating free DNA (cfDNA) from plasma in multiple myeloma (MM). : We used cell-free DNA from plasma and bone marrow to test , , and mutations using multiplex assays for droplet digital PCR (ddPCR), and evaluated results with clinical outcomes. : We found of 83 patients, the detectable mutation frequencies for the above four genes were 4 (5%), 13 (16%), 3 (4%) and 14 (17%) in bone marrow, respectively.

TERT, BRAF, and NRAS Mutational Heterogeneity between Paired Primary and Metastatic Melanoma Tumors.

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes.

Validation of Circulating Tumor DNA Assays for Detection of Metastatic Melanoma.

The detection of cell-free, circulating tumor DNA (ctDNA) in the blood of patients with solid tumors is often referred to as "liquid biopsy." ctDNA is particularly attractive as a candidate biomarker in the blood. It is relatively stable after blood collection, can be easily purified, and can be quantitatively measured with high sensitivity and specificity using advanced technologies.

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples.

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%).

Phasing DNA Markers Using Digital PCR.

Besides quantifying the absolute number of copies of known DNA targets, digital PCR can also be used to assess whether two nonpolymorphic gene sequences or two heterozygous markers reside on the same DNA molecule (i.e., are physically linked).

Very Low Abundance Single-Cell Transcript Quantification with 5-Plex ddPCR Assays.

Gene expression studies have provided one of the most accessible windows for understanding the molecular basis of cell and tissue phenotypes and how these change in response to stimuli. Current PCR-based and next generation sequencing methods offer great versatility in allowing the focused study of the roles of small numbers of genes or comprehensive profiling of the entire transcriptome of a sample at one time. Marrying of these approaches to various cell sorting technologies has recently enabled the profiling of expression in single cells, thereby increasing the resolution and sensitivity and strengthening the inferences from observed expression levels and changes.

Entering the Pantheon of 21 Century Molecular Biology Tools: A Perspective on Digital PCR.

After several decades of relatively modest use, in the last several years digital PCR (dPCR) has grown to become the new gold standard for nucleic acid quantification. This coincides with the commercial availability of scalable, affordable, and reproducible droplet-based dPCR platforms in the past five years and has led to its rapid dissemination into diverse research fields and testing applications. Among these, it has been adopted most vigorously into clinical oncology where it is beginning to be used for plasma genotyping in cancer patients undergoing treatment.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration.

Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci.

Sensitivity of plasma BRAFmutant and NRASmutant cell-free DNA assays to detect metastatic melanoma in patients with low RECIST scores and non-RECIST disease progression.

Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays.

A rapid molecular approach for chromosomal phasing.

Determining the chromosomal phase of pairs of sequence variants - the arrangement of specific alleles as haplotypes - is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well.

Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries.

Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples.

Unlabelled: The human epidermal growth factor receptor 2 (HER2, also known as erbB2) gene is involved in signal transduction for cell growth and differentiation. It is a cell surface receptor tyrosine kinase and a proto-oncogene. Overexpression of HER2 is of clinical relevance in breast cancer due to its prognostic value correlating elevated expression with worsening clinical outcome.

High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow.

High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays.

Background: A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets.

Analysis of molecular inversion probe performance for allele copy number determination.

We have developed a new protocol for using molecular inversion probes to accurately and specifically measure allele copy number. The new protocol provides for significant improvements, including the reduction of input DNA (from 2 mug) by more than 25-fold (to 75 ng total genomic DNA), higher overall precision resulting in one order of magnitude lower false positive rate, and greater dynamic range with accurate absolute copy number up to 60 copies.

Application of quantum dots to multicolor microarray experiments: four-color genotyping.

Highly multiplexed genomics assays are challenged by the need for a sufficient signal-to-noise ratio for each marker scored on a microarray-detection platform. Typically, as the number of markers scored (or target complexity) increases, either more assay-target material must be applied to the array or the specific activity of each marker must be proportionately increased. However, hybridization of excessive amounts of target to the microarray can result in elevated nonspecific binding and consequent degradation of information.

Allele quantification using molecular inversion probes (MIP).

Detection of genomic copy number changes has been an important research area, especially in cancer. Several high-throughput technologies have been developed to detect these changes. Features that are important for the utility of technologies assessing copy number changes include the ability to interrogate regions of interest at the desired density as well as the ability to differentiate the two homologs.

Multiplexed variation scanning for 1,000 amplicons in hundreds of patients using mismatch repair detection (MRD) on tag arrays.

Identification of the genetic basis of common disease may require comprehensive sequence analysis of coding regions and regulatory elements in patients and controls to find genetic effects caused by rare or heterogeneous mutations. In this study, we demonstrate how mismatch repair detection on tag arrays can be applied in a case-control study. Mismatch repair detection allows >1,000 amplicons to be screened for variations in a single laboratory reaction.

Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay.

Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of approximately 90% with concordance rates >99.

Genomic and physiological studies of early cryptochrome 1 action demonstrate roles for auxin and gibberellin in the control of hypocotyl growth by blue light.

Blue light inhibits elongation of etiolated Arabidopsis thaliana hypocotyls during the first 30 min of irradiation by a mechanism that depends on the phototropin 1 (phot1) photoreceptor. The cryptochrome 1 (cry1) photoreceptor begins to exert control after 30 min. To identify genes responsible for the cry1 phase of growth inhibition, mRNA expression profiles of cry1 and wild-type seedlings were compared using DNA microarrays.

Multiplexed genotyping with sequence-tagged molecular inversion probes.

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment.