RNA-Seq Truncation Site Preservation for Accurate Molecule Counting
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Solution Overview
Problem
Current RNA-seq methods for single-cell analysis often lose the link between 3' barcodes and transcript sequences during library preparation, leading to inaccurate counting of nucleic acid molecules due to biases and errors in amplification and sequencing processes.
Innovation Solution
A method involving random truncation of nucleic acid molecules at a specific base position, preserving the truncation site during amplification and sequencing, allowing for accurate alignment and counting of template molecules by maintaining unique truncation locations in the sequencing library.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If random truncation is performed on template nucleic acid molecules, then measurement precision of nucleic acid counting is improved, but device complexity of the sequencing system increases
Solution Approach 1:
The truncation site is established in advance during library preparation before sequencing. By pre-defining where the nucleic acid molecule should be truncated, the system creates a known reference point that simplifies subsequent alignment and counting operations, resolving the contradiction between precision and complexity
Solution Approach 2:
The truncation site acts as an intermediary marker between the template nucleic acid molecule and the sequencing read. This intermediate feature provides a reliable anchor point that enables accurate mapping without requiring complex computational methods, thus improving measurement precision while avoiding excessive system complexity
2Productivity
If amplification is performed on truncated nucleic acid molecules, then productivity of library preparation is improved, but loss of information about original molecule structure occurs
Solution Approach 1:
The method extracts only the essential information needed for counting - the truncation site position - while discarding unnecessary structural details of the original nucleic acid molecules. This selective extraction enables efficient amplification and sequencing while preserving only the critical counting information, resolving the contradiction between productivity and information retention
Solution Approach 2:
The nucleic acid molecule is segmented into functional regions: the truncation site (critical for counting), the amplifiable region (for productivity), and the sequencing read (for data collection). This segmentation allows independent optimization of each region's function, enabling high productivity while maintaining essential information
3Measurement precision
If truncation base positions are preserved during amplification, then measurement precision of transcript counting is improved, but manufacturing precision of library preparation increases
Solution Approach 1:
The method changes the parameter of nucleic acid length by introducing controlled truncation at specific base positions. This parameter change creates a standardized feature that can be reliably preserved through amplification, improving both measurement precision and manufacturing precision simultaneously by providing a consistent reference point across all library molecules
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 enables precise counting of nucleic acid molecules by ensuring that progeny polynucleotides retain the same truncation site, reducing errors and biases, and providing an absolute count of transcripts in the original sample, especially for low-input libraries from single cells.
Implementation Method 1
the truncating comprises performing base-catalyzed hydrolysis, ultrasonic shearing, or partial enzymatic degradation, of said plurality of template nucleic acid molecules
Implementation Method 2
the truncating comprises performing base-catalyzed hydrolysis, ultrasonic shearing, or partial enzymatic degradation, of said plurality of template nucleic acid molecules
Implementation Method 3
the truncating comprises performing base-catalyzed hydrolysis, ultrasonic shearing, or partial enzymatic degradation, of said plurality of template nucleic acid molecules
Data Source
AI summary
Disclosed herein are methods for counting nucleic acid molecules (e.g., RNA molecules) of a sample by randomly truncating the nucleic acid molecules at a truncation base position within the nucleic acid molecules to produce truncated nucleic acid molecules, amplifying and sequencing the truncated nucleic acid molecules to produce sequencing reads, aligning the sequencing reads to a reference sequence to produce aligned sequencing reads, and identifying a number of nucleic acid molecules using truncation locations of aligned sequencing reads. Also disclosed herein are methods for constructing sequencing libraries that preserve truncation positions of the nucleic acid molecules. Also disclosed herein are methods for depleting or enriching a sample for one or more target sequences, using sets of blocking oligonucleotides corresponding to the one or more target sequences.