Genomic-Scale Oligonucleotide Synthesis via TAB Sorting
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Solution Overview
Problem
Current technologies for synthesizing a complete or near-complete set of oligonucleotides representing a complex organism's genome are costly, with existing cell sorter accuracy resulting in significant sequence errors, making genome-scaled synthesis impractical due to high error rates and inefficiencies.
Innovation Solution
A combinatorial synthesizer utilizing multiple reaction vessels and advanced TAB sorters with a reader, sorting tree, and controller to accurately and quickly sort small, identifiable microtransponders (TABs) for precise oligonucleotide synthesis, enabling the recycling of ambiguously identified TABs and directing them back through the sorter for reassignment, thereby achieving high accuracy and efficiency in oligonucleotide production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If cell sorter technology is used to sort TABs, then sorting speed is achieved, but sorting accuracy is insufficient with a 1% error rate leading to sequence errors
Solution Approach 1:
The sorting process is divided into multiple sequential sorting passes, with each pass handling a specific subset of TABs or focusing on reducing error rates through repeated sorting operations. This segmentation allows the system to achieve both speed and accuracy by processing TABs in stages rather than attempting to sort all TABs in a single pass.
Solution Approach 2:
TABs are pre-sorted into groups before the main sorting operation, and ambiguous TABs are identified and re-sorted in subsequent passes. This preliminary organization reduces the complexity of the main sorting operation and enables more accurate sorting by reducing the number of decisions required at each sorting node.
2Manufacturing precision
If multiple sorting passes are implemented to improve accuracy, then sorting accuracy increases, but processing time increases
Solution Approach 1:
Different sorting strategies are applied to different subsets of TABs based on their identification confidence. TABs with clear, unambiguous identifiers are sorted in a single pass, while only TABs with ambiguous identifiers are subjected to multiple sorting passes. This localized application of multiple sorting passes minimizes the time penalty while maintaining high overall accuracy.
Solution Approach 2:
The sorting system uses digital records and tracking of TAB identities through multiple passes, allowing the system to remember and efficiently re-sort only the ambiguous TABs without re-processing already correctly sorted TABs. This copying of information about sorted TABs prevents redundant processing and reduces time loss.
3Reliability
If high accuracy sorting is achieved through multiple passes, then sequence error rate decreases, but system complexity increases
Solution Approach 1:
The system incorporates feedback mechanisms where the results of each sorting pass are analyzed to identify ambiguous TABs that require re-sorting. This feedback loop allows the system to adaptively focus subsequent sorting operations on only those TABs that need additional processing, maintaining high reliability without requiring complex hardware modifications.
Solution Approach 2:
A controller system acts as an intermediary between the sorting hardware and the decision-making process, managing the complexity of multiple sorting passes and ambiguous TAB identification. This intermediary layer simplifies the overall system architecture by centralizing the coordination of multiple sorting operations and the tracking of TAB identities.
Data Source
AI summary
Provided is a method of synthesis comprising: (I) providing separate reaction sequences to TABs; (II) utilizing reaction vessels configured to react a separate combinatorial building block with a moiety on a surface of a TAB; and (III) operating one or more TAB sorters comprising a TAB reader, a sorting tree comprising valves or switches and sorting nodes, and a monitor configured to detect TAB location, wherein the operating comprises serially conducting: (a) reacting distinct combinatorial building blocks in the reaction chambers with surfaces of TABs distributed in the reaction chambers; (b) operating a controller to operate the TAB sorters to segregate the TABs to allocations appropriate for the next assigned reaction, the operating including recycling TABs with ambiguous identity back through the sorter; and (c) repeating steps (a) and (b) as needed to complete 30% or more of the assigned sequences.