Bead-Bound DNA Library Retrieval via ID Sequencing
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Solution Overview
Problem
Current methods for synthetic DNA synthesis and recovery are labor-intensive, costly, and low-throughput, often resulting in unsuccessful syntheses that require repeating the entire process, with column-based phosphoramidite synthesis being error-prone and inefficient.
Innovation Solution
A method involving the synthesis of identifier regions on polynucleotide molecules, amplification, sequencing, and bead-bound library generation to accurately identify and retrieve error-free DNA sequences using massively parallel sequencing and targeted laser ejection, enabling high-throughput and cost-effective DNA synthesis by filtering out errors before assembly.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If column-based phosphoramidite synthesis is used for bulk-scale synthesis of oligonucleotides, then the synthesis process can be performed with standard equipment, but the process is error-prone, labor-intensive, and low-throughput
Solution Approach 1:
The patent segments the DNA synthesis process into two distinct stages: (1) bulk-scale synthesis of oligonucleotide mixtures using column-based phosphoramidite synthesis, and (2) selective amplification and sequencing of individual correct sequences using bead-based emulsion PCR. This segmentation allows each stage to be optimized independently, maintaining the ease of bulk synthesis while enabling high-throughput verification through parallel sequencing of multiple beads.
Solution Approach 2:
The patent creates multiple copies of individual correct oligonucleotide sequences by amplifying them on separate beads through emulsion PCR. Each bead serves as a template for generating numerous identical copies of the correct sequence, allowing parallel processing and sequencing of multiple correct molecules simultaneously, thereby dramatically increasing throughput without sacrificing accuracy.
2Ease of manufacture
If column-based phosphoramidite synthesis is used for bulk-scale synthesis of oligonucleotides, then the synthesis process can be performed with standard equipment, but the process is costly and requires repeating the entire process for unsuccessful syntheses
Solution Approach 1:
The patent performs preliminary sequencing of individual oligonucleotide sequences on beads before proceeding to assembly steps. By identifying and characterizing correct sequences early in the process, the system eliminates the need to repeat entire bulk synthesis processes for unsuccessful attempts, saving significant time and resources.
Solution Approach 2:
The patent implements feedback by sequencing the oligonucleotide sequences on beads and using this information to identify correct sequences for assembly. The sequencing results provide feedback that guides selective amplification and assembly, preventing wasted effort on incorrect sequences and reducing the need to repeat unsuccessful syntheses.
3Manufacturing precision
If individual sequence verification is performed after bulk-scale synthesis, then the accuracy of individual sequences can be confirmed, but the process becomes labor-intensive and low-throughput
Solution Approach 1:
The patent merges multiple individual sequencing operations into a single parallel process by amplifying and sequencing multiple oligonucleotide sequences simultaneously on different beads. This combining approach maintains high sequence accuracy through individual verification while achieving high throughput by processing many sequences in parallel.
Solution Approach 2:
The patent transitions from sequential individual sequence verification to parallel multi-dimensional sequencing by distributing multiple sequences across different beads and processing them simultaneously. This dimensional shift from one-dimensional sequential verification to multi-dimensional parallel processing dramatically increases throughput while maintaining accuracy.
4Productivity
If error-prone bulk synthesis is used, then the synthesis process can be performed efficiently in bulk, but errors in sequences reduce the quality of final DNA products
Solution Approach 1:
The patent extracts and isolates only the correct sequences from the bulk synthesis mixture by amplifying and sequencing individual beads, then selecting only those containing correct sequences for assembly. This extraction process removes errors from the final product while maintaining the efficiency of bulk synthesis through parallel processing.
Solution Approach 2:
The patent changes the parameter of sequence verification from post-synthesis individual checking to integrated bead-based parallel sequencing. By altering the verification parameter to occur simultaneously with amplification on beads, the system maintains high throughput while improving sequence accuracy through comprehensive parallel checking.
Data Source
AI summary
A method of retrieving a subset of polynucleotide molecules from a mixture of polynucleotide molecules includes receiving a mixture of nucleotide sequences comprising one or more polynucleotide molecules, synthesizing one or more identifier (ID) regions onto the one or more polynucleotide molecules, and sequencing members of the population of polynucleotide molecules to associate the sequence of one or more of the molecules (the “Polynucleotide Sequence”) with the sequence of the attached ID region (the “ID Sequence”). The method also includes generating a bead-bound library of one or more beads comprising subsets of identical polynucleotide molecules. Each bead is identified by the ID Sequence of the associated Polynucleotide Sequence. The method further includes sequencing the one or more ID regions of each bead to generate ID Sequence information for each bead, combining the Polynucleotide Sequence information, the one or more ID Sequences, and coordinates of each bead to identify the Polynucleotide Sequence on the bead, and retrieving the bead with its associated Polynucleotide Sequence from the flow cell based on the absolute coordinate position of the bead.