Immune Receptor Duplex Sequencing with Barcoded Strand Adapters
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Solution Overview
Problem
Existing methods for sequencing immune cell receptors, such as B cell and T cell receptors, face challenges with high error rates, particularly when detecting rare sequences, and are limited by biases and inefficiencies in library preparation, especially when dealing with low DNA amounts or small target regions.
Innovation Solution
A method involving attaching 3' and 5' adapter fragments with molecular barcodes to both strands of a double-stranded DNA molecule, followed by linear extension and separate sequencing of each strand, allowing for accurate determination of immune cell receptor sequences through duplex sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing sequencing methods are used, then sequencing can be performed, but error rate is too high to allow confident detection of rare sequences
Solution Approach 1:
The patent applies preliminary action by performing a first amplification step before sequencing to generate sufficient DNA copies from limited input samples. This pre-amplification ensures that rare sequences are adequately represented before the actual sequencing process, improving detection confidence without compromising accuracy
Solution Approach 2:
The patent uses molecular copying by generating multiple amplicons from each original DNA molecule through targeted amplification. This creates redundant copies that can be sequenced independently, allowing consensus calling to distinguish true rare sequences from sequencing errors, thereby improving both accuracy and reliability
2Quantity of substance
If DNA amount is limited, then sample availability is constrained, but sequencing coverage and accuracy decrease
Solution Approach 1:
The patent applies segmentation by dividing the sequencing process into targeted amplification of specific immune receptor regions followed by sequencing. This allows concentrated sequencing coverage on relevant regions from limited DNA, improving measurement precision without requiring increased total DNA quantity
Solution Approach 2:
The patent performs preliminary targeted amplification to enrich specific immune receptor sequences before sequencing. This pre-enrichment step maximizes the use of limited DNA by concentrating sequencing resources on relevant sequences, thereby maintaining high coverage and precision despite low input quantity
3Reliability
If complex library preparation methods are used, then sequencing capability is improved, but method complexity and cost increase
Solution Approach 1:
The patent applies universality by using a standardized targeted amplification approach that works across different immune receptor types and sequencing platforms. This multi-functional method simplifies library preparation while maintaining reliable sequencing capability, reducing both complexity and cost
Solution Approach 2:
The patent extracts and sequences only the relevant immune receptor regions through targeted amplification, rather than preparing libraries for entire genomes. This extraction approach simplifies the library preparation process while maintaining high sequencing capability for the specific application
Data Source
AI summary
Provided herein are methods for determining a sequence of a double stranded DNA molecule of an immune cell receptor (e.g., T cell receptor, B cell receptor).