Artificial RNA Reference Standards for Transcript Variant Quantification
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Solution Overview
Problem
Current methods for transcriptome analysis using short sequence reads face challenges in accurately aligning and quantifying transcript variants due to insufficient annotation, similarity between variants, and complexity in distinguishing variants expressed at similar levels.
Innovation Solution
A method involving a reference set of artificial nucleic acid molecules simulating transcript variants, with each family consisting of multiple molecules sharing exons and differing by significant sequences, is introduced. These molecules are added to samples as external controls for accurate identification and quantification through sequencing and detection methods like RNA-seq, micro-array analysis, or qPCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If short sequence reads are aligned to a reference genome or transcriptome, then transcriptome analysis can be performed, but accurate identification and quantification of transcript variants becomes difficult due to similarity between variants and insufficient annotation
Solution Approach 1:
The patent introduces artificial nucleic acid molecules as intermediary reference standards between the sequencing reads and the natural transcriptome. These synthetic molecules with known sequences and controlled abundances serve as mediators to calibrate the alignment process, providing a reference framework that resolves ambiguities in distinguishing similar transcript variants.
Solution Approach 2:
The patent employs parameter changes by systematically varying the abundance levels of different artificial transcript variants in the reference set. By creating reference molecules with known concentration gradients and spike-in at different levels, the method establishes quantitative parameters that improve the precision of transcript variant measurement and allow for accurate normalization.
2Quantity of substance
If multiple transcript variants are present in a sample, then biological complexity is captured, but reliable quantification becomes challenging due to variants expressed at similar levels and shared exonic sequences
Solution Approach 1:
The patent segments the reference transcriptome into multiple artificial gene families, each containing several transcript variants with defined relationships. By dividing the complex quantification problem into manageable gene-family units with controlled complexity, the method enables precise measurement of individual variants while maintaining the ability to detect multiple variants simultaneously.
Solution Approach 2:
The patent creates synthetic copies of transcript variants with known sequences and abundances. These artificial reference copies serve as templates for comparison, allowing the system to distinguish between natural transcript variants by comparing against controlled synthetic references that replicate the structural and sequence features of genuine transcripts.
3Productivity
If annotation is insufficient or differently curated, then genome mapping can proceed, but alignment accuracy of short reads to transcript variants deteriorates
Solution Approach 1:
The patent performs preliminary action by pre-construction of a comprehensive artificial reference transcriptome with fully annotated and curate d sequences before the actual sequencing experiment. This pre-established reference framework with known ground truth enables accurate alignment from the outset, eliminating the need to rely on insufficient or inconsistently curated existing annotations.
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 enhances the accuracy of transcript variant identification and quantification by providing controlled reference points, improving alignment and normalization, and allowing for reliable assessment of transcript variants in complex samples.
Implementation Method 1
at least one probe binds to at least one NA molecule of the reference set and a measuring result based on a signal resulting from the at least one probe binding to the at least one NA molecule
Data Source
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AI summary
The present invention relates to the field of transcriptomics and provides a method for the controlled identification and/or quantification of transcript variants in samples, comprising providing a reference set of artificial polynucleic acid molecules simulating transcript variants and adding said reference set as external control to samples comprising transcript variants. The present invention further provides such a reference set, as well as a method to produce such a reference set.