Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

Selective 2‘-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry allows local nucleotide flexibility to be quantitatively assessed at single nucleotide resolution in any RNA. SHAPE chemistry exploits structure-based gating of the nucleophilic reactivity of the ribose 2‘-hydroxyl group by the extent to which a nucleotide is constrained or flexible. SHAPE chemistry was developed using N-methylisatoic anhydride (NMIA), which is only moderately electrophilic and requires tens of minutes to form ribose 2‘-O-adducts. Here, we design and evaluate a significantly more useful, fast-acting, reagent for SHAPE chemistry. Introduction of a nitro group para to the reactive carbonyl to form 1-methyl-7-nitroisatoic anhydride (1M7) yields a reagent that both reacts significantly more rapidly with RNA to form 2‘-O-adducts and is also more labile toward advantageous, self-limiting, hydrolysis. With 1M7, the single nucleotide resolution interrogation of the RNA structure is complete in 70 s. SHAPE analysis performed with 1M7 accurately reports the secondary and tertiary structure of the RNase P specificity domain and allows the secondary structure of this RNA to be predicted with up to 91% accuracy.

A comprehensive understanding of RNA structure will provide fundamental insights into the cellular function of both coding and non-coding RNAs. Although many RNA structures have been analysed by traditional biophysical and biochemical methods, the low-throughput nature of these approaches has prevented investigation of the vast majority of cellular transcripts. Triggered by advances in sequencing technology, genome-wide approaches for probing the transcriptome are beginning to reveal how RNA structure affects each step of protein expression and RNA stability. In this Review, we discuss the emerging relationships between RNA structure and the regulation of gene expression.

New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPESeq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.chemical probing | RNA sequencing | RNA folding | genomics O ver the past several years, there has been an explosion in the discovery of noncoding, but functional RNAs that play central roles in maintaining, regulating, and defending the genome (1). At the same time, RNA-based mechanisms have emerged as powerful tools for engineering synthetic biological systems (2). Many of these natural and synthetic RNAs have specific secondary and tertiary structures essential to their function, and there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. Such a high-throughput structure characterization assay would allow rapid assessment of the impact of sequence on structure and function and enable RNA engineers to design libraries of RNA molecules with desired structural properties.Two techniques for high-throughput RNA structure characterization have recently been reported: parallel analysis of RNA structures (PARS) (3) and fragmentation sequencing (FragSeq) (4). Both techniques couple classic in vitro nuclease probing techniques that are traditionally performed one RNA at a time, with deep sequencing of RNA fragments to simultaneously probe a complex mixture of RNAs sampled from transcriptomes. Although important first steps, these techniques provide only low-resolution secondary structure information due to the limitations inherent in nuclease probing (5).We have developed a high-throughput technique, SHAPESeq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertia...