Michael Y. Lucero

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. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

A robust targeted sequencing approach for low input and variable quality DNA from clinical samples

Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification—this facilitates the accurate assessment of copy number alterations in addition to delivering accurate single nucleotide variant and insertion/deletion detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We demonstrate the performance using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types.Cancer diagnostics: Targeted DNA sequencing for low-quality tumor samplesA new DNA sequencing technology enables comprehensive genetic analyses of poor-quality tumor samples. Hanlee Ji from Stanford University in California, USA, together with colleagues from a company he cofounded called TOMA Biosciences, tested the performance of a targeted sequencing assay known as oligonucleotide-selective sequencing (OS-Seq). They used the “in-solution” version of OS-Seq, which involves a pre-processing step to remove any damaged DNA and then sequences target regions of the genome to look for duplications, insertions or deletions of DNA segments. Using archival specimens (which often contain low quantities of degraded DNA) from patients with lung and colorectal cancer, the researchers showed they could detect sequence variants in a panel of 130 cancer-related genes. The findings suggest the OS-Seq assay could help inform treatment decisions for cancer patients, even with clinical specimens of low quality.

Abstract 625: The frequency of gene amplifications in cancer revealed by a droplet digital PCR (ddPCR) based pan-cancer gene panel test

The development of molecular assays designed to detect gene amplifications has largely been hampered by technical challenges such as limited DNA quantity and tumor heterogeneity, which demand methods of very high precision and sensitivity. The recent introduction of affordable digital PCR platforms, such as droplet digital PCR (ddPCR), that are capable of providing single molecule resolution of target abundances thus provides a unique opportunity to address this gap in molecular diagnostics. A ddPCR-based test was therefore developed under CLIA-CAP guidelines to determine the amplification status of 12 commonly amplified genes targeted by FDA approved drugs. Termed the Amplinome Test, this test was applied to 49 clinical samples received over a period of 6 months and compared to the results obtained from a commercially available clinical sequencing test applied that also reports copy number alterations (CNAs). An overall concordance rate of 90% (532/588 calls) was observed between the two tests across the entire gene set, 5 of which were identified as amplified. There were 56 discordant copy number amplifications between the two testing modalities. The vast majority (55/56) of discordant calls arose from gene amplifications identified by the Amplinome test, but not detected by sequencing, indicating an 11-fold increase in sensitivity in detecting amplifications. One discordant call (HER3) was identified as amplified via sequencing, but confirmed to be unamplified via FISH. At the patient level, the Amplinome test identified 5-fold more patients as having actionable amplifications in at least one of the 12 assayed genes versus clinical sequencing (29 vs. 6). Clinical management was altered in 14 of the 29 patients with an actionable CNA identified on the Amplinome test; those patients received targeted therapy directed against the amplified gene. The ddPCR-based Amplinome test thus provides a highly sensitive method for measuring gene amplifications in cancer that alters patient management, and suggests that the prevalence of actionable amplifications may be significantly underestimated by standard clinical next-generation sequencing tests. Citation Format: Austin P. So, Amy Wong, Jennifer Pecson, Girish Putcha, Gregory Jensen, Michael Lucero, Gary Stone, Jason Gillman, Pravin Mishra, David Loughmiller, Derrick S. Haslem, Lincoln Nadauld. The frequency of gene amplifications in cancer revealed by a droplet digital PCR (ddPCR) based pan-cancer gene panel test. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2015-625