Asymmetric Barcoding for Rare Variant Detection
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Solution Overview
Problem
Current sequencing methods face challenges in accurately identifying rare sequence variants and copy number variations, particularly in limited patient samples, due to errors in sequencing and processing, and require improved accuracy to detect mutations at lower frequencies.
Innovation Solution
The method involves subjecting sense and antisense strand circular polynucleotides to rolling circle amplification with barcoded primers, followed by primer extension and sequencing to generate distinguishable amplicons, allowing for the identification of complementary strands and sequence variants based on unique barcodes and junction sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If next-generation sequencing is used to identify rare sequence variants, then sequencing depth and coverage are improved, but sequencing errors and processing errors increase false positive rates
Solution Approach 1:
The method segments the sequencing process by assigning unique molecular barcodes to individual DNA molecules before amplification. This allows tracking of each original molecule through multiple amplification cycles, enabling distinction between true variants and amplification errors. The segmentation principle is applied by dividing the sample into individually tracked molecular units rather than processing bulk DNA.
Solution Approach 2:
The patent uses molecular barcoding as a copying mechanism where each original DNA molecule is tagged with a unique identifier before amplification. The barcode serves as a digital copy that tracks the lineage of each molecule through PCR and sequencing, allowing error correction by comparing multiple copies derived from the same original molecule.
2Measurement precision
If the number of templates sequenced at each locus is increased to improve error analysis, then detection sensitivity is improved, but sample consumption increases
Solution Approach 1:
The method performs preliminary barcoding of individual DNA molecules before amplification. By tagging molecules in advance with unique identifiers, the system enables subsequent error analysis through computational methods that track barcode lineages, eliminating the need to physically increase template numbers for error detection.
Solution Approach 2:
The patent changes the parameter of molecular identification from sequence-based to barcode-based. This parameter change allows the system to track individual molecules through amplification without consuming additional sample, as the barcode provides a non-sequence identifier that can be computationally analyzed.
3Productivity
If rolling circle amplification is used to amplify circular polynucleotides, then amplification efficiency is improved, but formation of chimeric molecules increases
Solution Approach 1:
The method segments the amplification process by using unique barcodes on primers that bind to specific locations on circular templates. This allows tracking of each amplification event and identification of chimeric molecules through barcode analysis, enabling distinction between true amplification products and chimeric artifacts.
Solution Approach 2:
The patent implements feedback through computational analysis of barcode patterns in sequencing data. By analyzing the distribution and combination of barcodes, the system can identify chimeric molecules and exclude them from variant calling, providing feedback-based quality control.
4Measurement precision
If asymmetric barcoding with strand-specific primers is implemented, then ability to distinguish sense and antisense strands is improved, but primer design and reaction complexity increase
Solution Approach 1:
The method applies asymmetry by using different barcode sequences for sense and antisense strand primers. This asymmetric barcoding allows computational distinction between strands from complementary templates, enabling identification of true variants that appear on both strands versus artifacts on a single strand.
Solution Approach 2:
The patent creates universal barcoding primers that can bind to both sense and antisense circular templates while carrying strand-specific barcodes. This multi-functional primer design simplifies the overall process by using a single primer pair for both strands while maintaining strand identification capability through the barcode sequence.
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 detecting rare sequence variants and copy number variations by distinguishing between true sequence differences and errors, improving the sensitivity and specificity of mutation detection in limited sample sizes.
Implementation Method 1
subjecting a sense strand circular polynucleotide and an antisense strand circular polynucleotide originating from a double-stranded polynucleotide to a rolling circle amplification reaction using (i) a sense strand primer comprising a sense strand identifying barcode, and (ii) an antisense strand primer comprising an antisense strand identifying barcode
Implementation Method 2
the first set of auxiliary primers hybridize to a barcoded concatemer of the sense strand and the second set of auxiliary primers hybridize to a barcoded concatemer of the antisense strand
Implementation Method 3
subjecting the barcoded concatemers to a primer extension reaction using a first set and a second set of auxiliary primers to generate a plurality of amplicons
Implementation Method 4
sequencing the amplicons or derivatives thereof to produce sequencing reads
Data Source
AI summary
In some aspects, the present disclosure provides methods for identifying sequence variants, as well as methods of determining copy number of a genetic locus in a sample. Systems and kits for performing methods of the disclosure, as well as compositions produced by or useful in methods of the disclosure are also provided.