SBIR-STTR Award

This proposal addresses the problem of bias in the expression profiling of microRNAs (miRNAs) and other small RNAs by next-generation sequencing (NGS). Because dysregulation of miRNA expression has been implicated in cancer and certain other diseases, accurate expression profiling of all miRNA sequences is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. NGS is currently the most comprehensive approach for digital gene expression profiling, and is used for the discovery of novel miRNA sequences, identification of sequence variants, and the quantification of known miRNAs. Unlike other expression profiling platforms such as microarrays or RT-qPCR, NGS combines unlimited multiplexing capability, single-molecule sensitivity, a superior dynamic range, and true sequence specificity without requiring prior knowledge of miRNA sequences. However, NGS expression profiling data underestimate the amount of many miRNAs in a sample by as much as 10,000-fold. Knowledge of the absolute abundances in samples, and not just the relative changes between samples, is important for reliable identification of miRNAs as biomarkers or drug-target candidates. In NGS, the enzymatic ligation of adapters to the RNA ends is the step where most of the bias in miRNA quantification occurs. The major factors contributing to this ligation bias are intramolecular folding of the miRNAs and intermolecular folding between miRNAs and the adapters, which affect the ability of ligation enzymes to access and ligate the miRNA ends. Thus there is a need for new, more accurate methods, and most previous small RNA profiling experiments should be re- evaluated. To address these problems, we propose a new approach, miR-ACS (miRNA-Adapter Circularization and Sequencing), for preparing miRNA sequencing libraries. Key features of miR-ACS include (i) ligation of miRNAs with only a single adapter; (ii) circularizationof the miRNA-adapter ligation product; (iii) elimination of adapter species that are not ligated tomiRNAs; and (iv) RT-PCR amplification of the circular miRNA adapter adduct followed by gel-purification of amplicons suitable for NGS sequencing. MiR-ACS has the potential to essentially eliminate miRNA sequencing bias and significantly reduce the number of irrelevant miRNAs sequencing reads (allowing the sequencing of more samples in parallel). These features may also reduce cost and increase the throughput of NGS sequencing. Although miR-ACS is applicable to expression profiling of miRNA or other small RNAs in any sample, for proof-of-concept we focus on samples associated with prostate cancer.

Thesaurus Terms:
Address;Adduct;Affect;Applications Grants;Base;Biochemical Reaction;Bioinformatics;Biological Markers;Biology;Cancer Patient;Cancer Type;Clinical;Commercialization;Cost;Data;Development;Diagnostic;Digital;Disease;Drug Targeting;Enzymes;Excision;Gel;Gene Expression Profiling;Goals;Improved;Knowledge;Lead;Libraries;Ligase;Ligation;Malignant Neoplasm Of Prostate;Malignant Neoplasms;Methods;Micrornas;Molecular Profiling;Next Generation Sequencing;Novel;Novel Strategies;Patients;Phase;Plasma;Preparation;Procedures;Prostate;Protocols Documentation;Public Health Relevance;Reaction Time;Reading;Relative (Related Person);Research Study;Reverse Transcription;Rna;Rna Library;Rna Ligase (Atp);Rna Sequences;Rna-Directed Dna Polymerase;Sampling;Sequence Analysis;Services;Single Molecule;Small Rna;Solute;Specificity;Temperature;Therapeutic Target;Tissue Sample;Tool;Variant;

This proposal addresses the problem of sequence bias in next-generation sequencing (NGS) of small RNAs such as microRNAs (miRNAs) as well as fragments of larger RNAs. Because dysregulation of miRNA expression has been implicated in cancer and other diseases, accurate expression profiling of all miRNA sequences is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. NGS is currently the most comprehensive approach for discovery and expression profiling of small RNA sequences. However, NGS expression profiling data underestimate the abundance of most miRNAs in a sample, some by as much as 10,000-fold. Knowledge of the true abundances in samples, and not just the relative changes between samples, is important for reliable identification of miRNAs as biomarkers or drug-target candidates. Other advantages of unbiased detection include the ability to discover novel RNAs and detect low-abundance RNAs that cannot be detected by current NGS methods, especially in samples with a low concentration of RNA. The source of bias in currently available methods of preparation RNA sequencing libraries for NGS is inefficient and sequence-dependent ligation of the two sequencing adapters to the sample RNAs. The major factors contributing to this ligation bias are intramolecular folding of the miRNAs and intermolecular folding between miRNAs and adapters, which affect the ability of the ligase to access and ligate the miRNA ends. Thus there is a need for new, more accurate methods, and most previous small RNA profiling experiments should be re-evaluated. To address these problems, we are developing a new approach, miR- ACS (miRNA-Adapter Circularization and Sequencing), for preparing unbiased sequencing libraries that is applicable to miRNAs and other small RNAs as well as small fragments of large RNAs used in general RNA- Seq. Key features of miR-ACS include (i) ligation of miRNAs with only a single combo adapter (CAD) that combines sequences of the standard 3'- and 5'-adapters used for Illumina sequencing, producing miRNA-CAD ligation products; (ii) circularization of the miRNA-CAD products; (iii) blocking of free CAD species that are not ligated to miRNAs; and (iv) RT-PCR amplification of the circular miRNA-CAD products to produce standard sequencing amplicons containing a single RNA-specific sequence insert flanked by the 5'- and 3'-adapter sequences. In Phase I, we have demonstrated the feasibility of the miR-ACS approach (proof-of-concept) by greatly reducing the miRNA sequencing bias in comparison to the best current library prep methods. In Phase II we will thoroughly optimize miR-ACS to maximize bias reduction and to allow sequencing of a larger variety of RNAs (up to 150 nt in size) with very low RNA inputs. In addition, we will streamline the protocol to facilitate its adoption by users and for commercial viability.

Public Health Relevance Statement:
HEALTH RELATEDNESS NARRATIVE MicroRNAs (miRNAs) are promising candidates as biomarkers of cancer and other diseases that could allow early diagnosis, sub-typing, choice of targeted treatments, and monitoring of the progress of therapy. Although, next-generation sequencing (NGS) is the method of choice for discovering and expression profiling of miRNAs, it underestimates the abundance of most miRNAs, some by as much as 10,000-fold because of sequence bias. The goal of this grant application is to eliminate this bias to allow discovery of new RNA species and determination of the true abundance of RNAs in biological samples.

Project Terms:
Address; Adoption; Affect; Applications Grants; Biological; Biological Markers; Biology; cancer biomarkers; Cancer Patient; cost; Data; design; Detection; Development; dimer; Disease; Drug Targeting; Early Diagnosis; Gel; Goals; Health; human tissue; Individual; Knowledge; Letters; Libraries; Ligase; Ligation; Malignant Neoplasms; Measurement; Messenger RNA; Methods; microRNA biomarkers; MicroRNAs; Molecular Profiling; Monitor; New England; next generation sequencing; novel; novel strategies; Performance; Phase; Phosphorylation; Preparation; prevent; Procedures; Progress Reports; Protocols documentation; prototype; Publications; Reaction; Reading; Reagent; Reporting; Reproducibility; research study; Reverse Transcription; RNA; RNA Sequences; Role; Sampling; Small RNA; Source; Technology; Testing; therapeutic target; Time; Transcript; transcriptome sequencing; tumor; Work