Dual Barcode Insertion for Accurate NGS Read Alignment
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Solution Overview
Problem
Next-generation sequencing (NGS) technologies face challenges in accurately aligning sequence reads due to errors and complexities, leading to incorrect variant detection and ambiguous sequencing results, particularly in determining the sequence of target polynucleotides with repeat regions or multiple isoforms.
Innovation Solution
Insertion of polynucleotide barcodes into target polynucleotides using probes with insertion vectors like CRISPR-Cas9 or Tn5 transposase, followed by sequencing and removing the barcodes to unambiguously determine the sequence, improving alignment and variant detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If NGS technologies are used to sequence DNA fragments in parallel, then sequencing speed and productivity are improved, but alignment accuracy deteriorates due to errors and complexities in the read process
Solution Approach 1:
The patent introduces barcode sequences as intermediary elements that are inserted into the target polynucleotide before sequencing. These barcodes serve as mediators between the sequencing process and the alignment process, providing reference points that enable accurate alignment of reads even when sequencing errors occur. The barcodes act as a common reference framework that resolves ambiguities in read alignment.
Solution Approach 2:
The patent applies preliminary action by inserting barcode sequences into the target polynucleotide before the actual sequencing process. This preliminary modification ensures that alignment reference points are already in place, allowing the sequencing machine to generate reads that can be accurately aligned later, even though the sequencing itself may contain errors.
2Productivity
If computational techniques are used to align sequence reads, then sequencing throughput is improved, but reliability deteriorates due to misalignment and incorrect variant detection
Solution Approach 1:
The barcode sequences serve as reliable intermediary reference points that computational algorithms can use to accurately align reads. Instead of relying solely on complex algorithms to resolve ambiguities, the barcodes provide concrete, unambiguous reference positions that improve the reliability of automated alignment and variant detection processes.
Solution Approach 2:
The barcode sequences provide feedback mechanisms for alignment accuracy. By including known barcode sequences in the target polynucleotide, the system enables verification of alignment correctness through the presence or absence of expected barcode patterns in the sequencing reads, allowing for feedback-based correction of misalignments.
3Adaptability or versatility
If read fragments are aligned relative to each other, then assembly capability is improved, but measurement precision deteriorates due to overlapping fragments and alignment ambiguities
Solution Approach 1:
The barcode sequences act as intermediary anchors that resolve ambiguities in fragment assembly. When multiple reads overlap, the barcode sequences provide unique identification and positioning information that enables precise determination of fragment relationships, eliminating ambiguities that would otherwise exist in purely relative alignment approaches.
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
Enhances the accuracy of sequencing by providing additional information for fragment alignment and variant detection, reducing ambiguity in sequencing results, especially in repeat regions and multiple isoforms.
Implementation Method 1
the insertion vector inserts the payload polynucleotide into the target polynucleotide
Implementation Method 2
using probes with insertion vectors like CRISPR-Cas9 or Tn5 transposase
Implementation Method 3
applying an amplification agent to generate: (1) a first copy of the target polynucleotide, with the payload polynucleotide inserted
Data Source
AI summary
Contemporary gene sequencing techniques, including “Next Generation Sequencing” techniques, can include sequencing a plurality of fragments of a target polynucleotide. However, tire limitations of existing sequencing techniques, and the often repetitive or otherwise difficult-to-sequence structure of natural polynucleotides, means that it can be difficult and/or expensive to generate accurate sequences. Methods provided herein include inserting dual polynucleotide ‘barcodes,’ along with neighboring primer sequences, into a target polynucleotide prior to other sequencing processes. These inserted barcodes can improve the accuracy of sequences generated for the target by adding ‘noise’ into the target, allowing subsequent sequencing techniques (e.g., alignment, stitching, etc.) to more accurately estimate the target-plus-barcodes sequence. The primers can cause the fragments to begin at points within the target that correspond to the beginning of other sequences, facilitating the stitching of sequence ends together. The barcodes can then be removed to provide the sequence of the target polynucleotide.