Barcoded Circular Library Construction for Chimeric Product Identification
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Solution Overview
Problem
Current nucleic acid sequencing methods using barcodes struggle to accurately detect low-frequency gene fusions due to the random ligation of barcodes, which makes it difficult to distinguish between authentic and artificially produced chimeric molecules during amplification, limiting the detection of true mutations in cancer.
Innovation Solution
A method involving the ligation of a single adaptor with two barcodes and primer binding sites to target molecules, forming circular molecules, where modified nucleotides terminate strand synthesis, allowing for precise barcode placement and differentiation between authentic and artificial gene fusions through sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If barcodes are randomly ligated to both ends of target molecules, then barcode coverage is achieved, but chimeric molecules are produced via template switching in PCR making it impossible to distinguish authentic gene fusions from artifacts
Solution Approach 1:
The adaptor is segmented into distinct functional regions: a first barcode region, a primer binding site, a second barcode region, and a modified nucleotide. This segmentation allows the first barcode to be exclusively associated with the 5' end and the second barcode with the 3' end of the target molecule, preventing random ligation and enabling accurate identification of authentic gene fusions versus PCR artifacts.
Solution Approach 2:
The adaptor is designed with a modified nucleotide that effects strand synthesis termination by a nucleic acid polymerase. This preliminary structural feature prevents the polymerase from extending through the adaptor sequence during PCR, thereby preventing template switching and the formation of chimeric molecules before they can occur.
2Measurement precision
If barcodes are used for error correction, then sensitivity for detecting rare point mutations is enhanced, but the same barcode structure cannot differentiate authentic translocations from artifacts
Solution Approach 1:
Different regions of the adaptor structure serve different functions: the first barcode region is optimized for identifying the 5' end origin, the second barcode region identifies the 3' end origin, and the modified nucleotide provides local termination quality. This local differentiation allows simultaneous achievement of high sensitivity through barcode error correction and high specificity through authentic chimeric molecule identification.
3Manufacturing precision
If a single adaptor with two barcodes is ligated to form circular molecules, then precise barcode placement is achieved, but the ligation process complexity increases
Solution Approach 1:
The single adaptor design serves multiple functions simultaneously: it provides two barcodes for endpoint identification, contains primer binding sites for amplification, includes a modified nucleotide for strand termination, and enables circularization of the target molecule. This multi-functionality reduces the need for multiple separate reagents and simplifies the overall workflow despite the increased molecular complexity of the adaptor itself.
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 ability to detect true gene fusions by eliminating artifacts and improving the sensitivity and specificity of mutation detection, enabling the identification of important driver mutations in cancer.
Implementation Method 1
at least one modified nucleotide effecting a strand synthesis termination by a nucleic acid polymerase situated between the two primer binding sites
Implementation Method 2
annealing a forward primer complementary to the adaptor to one strand of the target molecule; annealing a reverse primer complementary to the adaptor to the first strand
Implementation Method 3
extending the forward primer up to the modified nucleotide, thereby producing a first strand; extending the first primer, thereby producing the second strand
Implementation Method 4
ligating a single adaptor to a target molecule forming a circular molecule
Data Source
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AI summary
The invention is a novel method of constructing libraries for single-molecule sequencing and a composition therefor. The method utilizes barcodes that enable detection and sequencing of chimeric target molecules with great sensitivity. The method finds application in detection gene fusions such as the ones characteristic of cancer.