Asymmetric DNA Tagmentation Adapter for NGS Library Preparation
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Solution Overview
Problem
Current next-generation sequencing (NGS) methods face challenges in efficiently preparing libraries for whole-genome sequencing, particularly due to the requirement for different tags on both ends of DNA fragments post-tagmentation, leading to loss of fragments with the same sequences, and the difficulty in distinguishing between sequencing errors and true mutations, especially in samples with low copy numbers of mutant molecules.
Innovation Solution
A method involving an adapter comprising oligonucleotides that are hybridized to produce a complex with a transposase recognition sequence, a central single-stranded region of variable sequence, and non-complementary ends, allowing for tagging of both strands of DNA fragments with the same molecular barcode during tagmentation, which enhances the identification of true mutations by ensuring that sequence variations are present on both strands.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional tagmentation methods are used with two different tags on transposase, then PCR amplification can proceed, but 50% of fragments are lost when both ends have the same sequence
Solution Approach 1:
The patent uses asymmetric tagmentation where only one end of the DNA fragment receives a tag during transposase insertion. This asymmetric tagging prevents the 50% fragment loss problem because all fragments have at least one tagged end, eliminating the requirement for both ends to have different tags for PCR amplification.
Solution Approach 2:
Instead of requiring two different tags on both ends of fragments (conventional approach), the patent inverts the strategy by using a single tag type on one end only. This inversion of the tagging strategy simplifies the process and eliminates fragment loss while maintaining PCR amplification capability.
2Ease of operation
If multiple different tags are loaded onto transposase to ensure different sequences on fragment ends, then PCR amplification is enabled, but device complexity and procedure difficulty increase
Solution Approach 1:
The patent employs a universal tagging approach where a single tag sequence serves multiple functions: it enables PCR amplification, provides sequencing adaptors, and works for all fragments regardless of their origin. This universal tag eliminates the need for multiple different tags, simplifying both the transposase loading process and overall procedure.
Solution Approach 2:
The patent uses homogeneous tagging where all fragments receive the same tag sequence on the tagged end. This homogeneity simplifies the transposase loading process (only one tag type needed) and facilitates uniform PCR amplification and sequencing procedures, reducing operational complexity compared to heterogeneous multi-tag approaches.
3Measurement precision
If traditional sequencing methods are used, then sequencing can be performed, but it is impossible to distinguish between sequencing errors and true mutations in low copy number samples
Solution Approach 1:
The patent uses molecular barcoding where each original DNA molecule is copied with a unique identifier tag. During PCR amplification and sequencing, this barcode is replicated along with the DNA sequence. By tracking which sequences share the same barcode, the system can distinguish true mutations (present in multiple copies with same barcode) from sequencing errors (appearing in only one or a few copies).
Solution Approach 2:
The patent implements feedback through barcode tracking, where the sequence data is fed back through the barcode identifier to verify authenticity. If a potential mutation is detected, the system checks whether it appears in multiple sequences sharing the same molecular barcode, providing feedback confirmation that distinguishes true mutations from errors.
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 improves the capture and tagging of low-copy number mutations, reducing the loss of fragments and increasing confidence in identifying true sequence variations by ensuring that mutations are present on both strands of the DNA, thereby improving the accuracy of sequencing results.
Implementation Method 1
genomic DNA can be prepared for next-generation sequencing (NGS) by 'tagmentation', where the transposase causes staggered double-stranded breaks in the genomic DNA and simultaneously inserts small oligonucleotide tags on the ends
Implementation Method 2
an adapter comprising a population of first oligonucleotides, a second oligonucleotide and a third oligonucleotide, wherein the first oligonucleotides, the second oligonucleotide and the third oligonucleotide are hybridized together to produce a complex
Implementation Method 3
filling in and sealing the central single-stranded region of the adaptor using a polymerase
Implementation Method 4
sealing the central single-stranded region of the adaptor using a polymerase and ligase
Data Source
AI summary
Described herein is an adapter comprising a population of first oligonucleotides, a second oligonucleotide and a third oligonucleotide, wherein the first oligonucleotides, the second oligonucleotide and the third oligonucleotide are hybridized together to produce a complex that comprises: (i) a first end comprising a transposase recognition sequence, (ii) a central single-stranded region of variable sequence and (iii) a second end comprising sequences that are non-complementary. A method, as well as a kit for practicing the method, are also provided.