De Novo DNA Sequencing Using CID and Radical Dissociation
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Solution Overview
Problem
Existing de novo sequencing methods, such as those described in the Gabelica Paper, are limited by base-dependency and lack applicability to dissociation techniques other than electron photodetachment dissociation (EPD), necessitating the development of systems and methods that overcome these limitations and are applicable to multiple dissociation techniques.
Innovation Solution
The method involves de novo sequencing using both thermal-dissociation (e.g., CID) and radical-induced dissociation (e.g., plasma EDD) techniques, analyzing nucleic acids through a· and w ion series fragments, and employing charge state deconvolution to identify nucleotides by mass differences between spectra from these methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If electron photodetachment dissociation (EPD) is used for de novo sequencing, then sequencing can be performed, but the method is limited by base-dependency and requires guanines to be present for EPD to occur
Solution Approach 1:
The patent combines two different dissociation methods (EPD and CID) into a single sequencing workflow. By merging the spectral information from both methods, the system overcomes the base-dependency limitation of EPD alone, as CID can dissociate nucleic acids regardless of base composition, particularly when guanines are absent.
Solution Approach 2:
The patent creates a universal sequencing method that works across different base compositions by making the overall process multi-functional. The system can handle sequences with or without guanines by switching between or combining EPD and CID, making the sequencing approach adaptable to various nucleic acid compositions rather than being restricted to specific base patterns.
2Device complexity
If a single dissociation method is used for sequencing, then the process is simpler, but the method lacks applicability to various dissociation techniques and has limited versatility
Solution Approach 1:
The patent develops a universal sequencing framework that can accommodate multiple dissociation methods (EPD, CID, and potentially others). The system is designed to process and integrate data from different dissociation techniques, making it versatile and adaptable to various methods rather than being locked into a single approach.
Solution Approach 2:
The patent segments the sequencing process into distinct analytical stages that can handle different types of spectral data. By dividing the overall sequencing workflow into manageable segments that can process EPD spectra, CID spectra, or combinations thereof, the system maintains relative simplicity while achieving broad applicability across multiple dissociation techniques.
3Loss of information
If de novo sequencing is performed without additional information, then true de novo sequencing is achieved, but the accuracy and reliability may be compromised without reference data
Solution Approach 1:
The patent merges spectral information from multiple dissociation methods to compensate for the lack of additional reference information. By combining the complementary data from EPD and CID spectra, the system achieves sufficient information content to perform accurate de novo sequencing without relying on genomic databases or pre-obtained sequence information.
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 allows for accurate reconstruction of nucleic acid sequences without additional information, overcoming base-dependency and expanding applicability to various dissociation techniques, thereby enhancing sequencing efficiency and accuracy.
Implementation Method 1
a first product ion mass spectrum of a nucleic acid analyzed using a thermal-dissociation method is received
Implementation Method 2
a second product ion mass spectrum of the nucleic acid analyzed using a radical-induced dissociation method is received
Data Source
AI summary
A first product ion mass spectrum of a nucleic acid analyzed using a CID method is received. Also, a second product ion mass spectrum of the nucleic acid analyzed using a radical-induced dissociation method is received. Peak m/z values of the first spectrum, peak m/z values of the second spectrum, and an m/z value of a precursor ion of the nucleic acid are converted to a single charge. A peak m/z value of the first spectrum is determined that differs from a peak m/z value of the second spectrum by a mass value of a structure within the nucleic acid the radical-induced dissociation method is known to not be able to dissociate and the CID method is known to be able to dissociate. The structure includes a phosphorus atom and an optionally substituted 5-membered ring containing an oxygen.