Semi-Random Barcodes for Error-Corrected Nucleic Acid Sequencing
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Solution Overview
Problem
Next generation sequencing (NGS) technologies face errors in mutation detection and transcriptome profiling due to sequence-dependent bias and amplification noise, making it difficult to accurately identify rare mutations and count transcripts.
Innovation Solution
The use of semi-random barcodes to tag sequencing fragments before amplification, allowing for error correction and reducing bias and sequencing errors through oligonucleotides with semi-random barcode sequences in sequencing adapters, reverse transcription primers, and PCR primers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a random sequence barcode is used to tag template molecules, then amplification bias and sequencing errors can be corrected, but the barcode can be frequently misidentified due to sequencing errors in the barcode region
Solution Approach 1:
The patent applies local quality by creating barcodes with different error characteristics in different regions. The first barcode region has higher error tolerance while the second barcode region has lower error tolerance, allowing the system to correct errors locally in the first region while maintaining precision in the second region for final identification.
Solution Approach 2:
The patent segments the barcode into two distinct parts: a first barcode region with higher error rate tolerance for correcting amplification bias, and a second barcode region with lower error rate for accurate identification. This segmentation allows each region to serve its specific function optimally.
2Reliability
If a predefined sequence barcode is used to tag template molecules, then sequencing errors can be corrected, but the cost for generating large varieties of barcodes is very high
Solution Approach 1:
The patent changes the parameter of barcode sequence randomness from completely random or completely predefined to semi-random. This allows the system to maintain error correction capabilities while reducing the cost of generating diverse barcodes by using a controlled randomization approach rather than synthesizing every possible predefined sequence.
3Productivity
If next generation sequencing technology is used for mutation detection and transcriptome profiling, then high throughput sequencing is achieved, but the error rate prevents confident identification of rare mutations
Solution Approach 1:
The patent implements feedback by using the first barcode region to detect and correct errors that occur during amplification and sequencing. The error correction information from the first region is fed back to correct the second region, improving the overall accuracy of mutation detection while maintaining high throughput.
Data Source
AI summary
The present disclosure provides oligonucleotides that comprise semi-random barcode sequences. Such oligonucleotides may be incorporated into reverse transcription primers, PCR primers, or portions of sequencing adapters in preparing sequencing libraries. The resulting sequencing libraries can be used for accurate sequencing, including DNA or RNA counting and mutation detection. Methods and kits for preparing sequencing adapters and sequencing libraries are also provided.