Small RNA Sequencing via Ribosomal Masking and Template Switching
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Solution Overview
Problem
Current methods for sequencing short RNAs are hindered by the need for large amounts of starting material, contamination from ribosomal RNA, and biases in sample preparation, making it difficult to achieve sensitive and reliable quantification, especially for single cells or complex tissues.
Innovation Solution
A method involving masking ribosomal RNA, ligating adaptors to small RNA molecules, and performing reverse transcription and PCR to enable sequencing and quantification without size selection, suitable for single cell analysis and automation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If gel-based assays, microarrays, or real-time PCR are used for sRNA profiling, then detection of known RNA sequences is possible, but target amplification is difficult due to the short size of sRNAs and high input target concentration is required
Solution Approach 1:
The patent performs size selection of sRNAs before the main sequencing workflow, and uses template switching oligos with specific sequences designed to facilitate efficient reverse transcription and PCR amplification of short RNA molecules. These preliminary preparations address the amplification difficulty inherent to short sRNAs.
Solution Approach 2:
The patent introduces template switching oligos as intermediaries that bridge the gap between short sRNA molecules and the sequencing library preparation workflow. These oligos contain sequences that facilitate efficient binding and extension, enabling amplification of short targets that would otherwise be difficult to amplify.
2Productivity
If next generation sequencing-based sRNA profiling is performed, then high throughput and comprehensive sequencing is achieved, but much larger amounts of starting material are required compared to longer RNA fragments
Solution Approach 1:
The patent performs size selection to enrich for sRNAs of specific lengths (18-30 nucleotides) before library preparation and sequencing. This preliminary enrichment step increases the proportion of target molecules in the sample, enabling efficient sequencing with limited starting material.
Solution Approach 2:
The patent optimizes various parameters including the size selection range, adapter ligation conditions, and PCR cycling parameters to maximize the efficiency of library preparation from minimal sRNA input while maintaining high sequencing throughput.
3Manufacturing precision
If size selection steps are included in sRNA sequencing protocols, then sequencing accuracy is improved, but implementation complexity and time consumption increase
Solution Approach 1:
The patent performs size selection as a preliminary step using gel electrophoresis or size-exclusion chromatography to separate sRNAs by length. This preliminary purification ensures that only the desired size range proceeds to library preparation, improving sequencing accuracy while establishing a clear workflow structure.
4Quantity of substance
If bulk RNA analysis is performed instead of single cell analysis, then sufficient RNA quantity is available for sequencing, but resolution of RNA expression on single cell level is lost
Solution Approach 1:
The patent divides the sample into individual single cells and processes each cell separately through the sRNA sequencing workflow. This segmentation enables measurement of sRNA expression at single-cell resolution while the optimized protocol ensures sufficient RNA is captured and processed from each individual cell.
Solution Approach 2:
The patent uses whole transcriptome amplification and template switching oligos as intermediaries to amplify the limited RNA content from single cells to levels sufficient for sequencing, while preserving the single-cell identity through barcoding or separate processing.
5Ease of manufacture
If traditional sRNA profiling methods are used, then established protocols are available, but detection of rare targets and post-assay validation with more sensitive assays is required
Solution Approach 1:
The patent performs size selection and enrichment of sRNAs before sequencing, and uses optimized adapter ligation and PCR conditions to preferentially amplify rare targets. This preliminary enrichment improves the detection sensitivity for rare sRNA species without requiring post-assay validation.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for high-sensitivity, reproducible, and quantitative analysis of short RNA sequences from small amounts of material, reducing contamination effects and biases, and facilitating the detection of rare RNA species in complex mixtures.
Implementation Method 1
masking ribosomal RNA molecules by providing one or more oligonucleotide(s) complementary to the RNA to be masked
Implementation Method 2
ligating a 3' oligonucleotide adaptor to the 3' end of small RNA molecules
Implementation Method 3
performing reverse transcription of the RNA
Data Source
AI summary
Methods and kits for sequencing and quantification of small RNA molecules from small quantities of starting materials, including single cells, are disclosed. The invention can be applied to, among others, preparing small RNA libraries, synthesizing cDNA from small RNAs, characterizing small RNAs from various cell types, studying cellular heterogeneity in pathological conditions, and diagnosing diseases.