skip to main content

Title: Ultra-rapid somatic variant detection via real-time targeted amplicon sequencing

Molecular markers are essential for cancer diagnosis, clinical trial enrollment, and some surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform due to time-consuming DNA extraction, targeted amplification, and library preparation times. In this work, we present a proof-of-principle approach for sub-1 hour targeted variant detection using real-time DNA sequencers. By modifying existing protocols, optimizing for diagnostic time-to-result, we demonstrate confirmation of a hot-spot mutation from tumor tissue in ~52 minutes. To further reduce time, we explore rapid, targeted Loop-mediated Isothermal Amplification (LAMP) and design a bioinformatics tool—LAMPrey—to process sequenced LAMP product. LAMPrey’s concatemer aware alignment algorithm is designed to maximize recovery of diagnostically relevant information leading to a more rapid detection versus standard read alignment approaches. Using LAMPrey, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes.

; ; ; ; ; ; ; ; ;
Award ID(s):
Publication Date:
Journal Name:
Communications Biology
Nature Publishing Group
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract BACKGROUND

    Despite widespread interest in next-generation sequencing (NGS), the adoption of personalized clinical genomics and mutation profiling of cancer specimens is lagging, in part because of technical limitations. Tumors are genetically heterogeneous and often contain normal/stromal cells, features that lead to low-abundance somatic mutations that generate ambiguous results or reside below NGS detection limits, thus hindering the clinical sensitivity/specificity standards of mutation calling. We applied COLD-PCR (coamplification at lower denaturation temperature PCR), a PCR methodology that selectively enriches variants, to improve the detection of unknown mutations before NGS-based amplicon resequencing.


    We used both COLD-PCR and conventional PCR (for comparison) to amplify serially diluted mutation-containing cell-line DNA diluted into wild-type DNA, as well as DNA from lung adenocarcinoma and colorectal cancer samples. After amplification of TP53 (tumor protein p53), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), IDH1 [isocitrate dehydrogenase 1 (NADP+), soluble], and EGFR (epidermal growth factor receptor) gene regions, PCR products were pooled for library preparation, bar-coded, and sequenced on the Illumina HiSeq 2000.


    In agreement with recent findings, sequencing errors by conventional targeted-amplicon approaches dictated a mutation-detection limit of approximately 1%–2%. Conversely, COLD-PCR amplicons enriched mutations above the error-related noise, enabling reliable identification of mutation abundances of approximatelymore »0.04%. Sequencing depth was not a large factor in the identification of COLD-PCR–enriched mutations. For the clinical samples, several missense mutations were not called with conventional amplicons, yet they were clearly detectable with COLD-PCR amplicons. Tumor heterogeneity for the TP53 gene was apparent.


    As cancer care shifts toward personalized intervention based on each patient's unique genetic abnormalities and tumor genome, we anticipate that COLD-PCR combined with NGS will elucidate the role of mutations in tumor progression, enabling NGS-based analysis of diverse clinical specimens within clinical practice.

    « less
  2. Abstract Thus far immunotherapy has had limited impact on ovarian cancer. Vigil (a novel DNA-based multifunctional immune-therapeutic) has shown clinical benefit to prolong relapse-free survival (RFS) and overall survival (OS) in the BRCA wild type and HRP populations. We further analyzed molecular signals related to sensitivity of Vigil treatment. Tissue from patients enrolled in the randomized double-blind trial of Vigil vs. placebo as maintenance in frontline management of advanced resectable ovarian cancer underwent DNA polymorphism analysis. Data was generated from a 981 gene panel to determine the tumor mutation burden and classify variants using Ingenuity Variant Analysis software (Qiagen) or NIH ClinVar. Only variants classified as pathogenic or likely pathogenic were included. STRING application (version 1.5.1) was used to create a protein-protein interaction network. Topological distance and probability of co-mutation were used to calculated the C-score and cumulative C-score (cumC-score). Kaplan–Meier analysis was used to determine the relationship between gene pairs with a high cumC-score and clinical parameters. Improved relapse free survival in Vigil treated patients was found for the TP53 m- BRCA wt-HRP group compared to placebo (21.1 months versus 5.6 months p  = 0.0013). Analysis of tumor mutation burden did not reveal statistical benefit in patients receiving Vigil versusmore »placebo. Results suggest a subset of ovarian cancer patients with enhanced susceptibility to Vigil immunotherapy. The hypothesis-generating data presented invites a validation study of Vigil in target identified populations, and supports clinical consideration of STRING-generated network application to biomarker characterization with other cancer patients targeted with Vigil.« less
  3. Rasmussen, Angela L. (Ed.)
    ABSTRACT Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accuratemore »and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.« less
  4. Abstract

    Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65 °C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity.

  5. Due to the recent outbreak of the Zika virus (ZIKV) in several regions, rapid, and accurate methods to diagnose Zika infection are in demand, particularly in regions that are on the frontline of a ZIKV outbreak. In this paper, three diagnostic methods for ZIKV are considered. Viral isolation is the gold standard for detection; this approach can involve incubation of cell cultures. Serological identification is based on the interactions between viral antigens and immunoglobulin G or immunoglobulin M antibodies; cross-reactivity with other types of flaviviruses can cause reduced specificity with this approach. Molecular confirmation, such as reverse transcription polymerase chain reaction (RT–PCR), involves reverse transcription of RNA and amplification of DNA. Quantitative analysis based on real-time RT–PCR can be undertaken by comparing fluorescence measurements against previously developed standards. A recently developed programmable paper-based detection approach can provide low-cost and rapid analysis. These viral identification and viral genetic analysis approaches play crucial roles in understanding the transmission of ZIKV.