Nucleic Acid Analysis With Barcoded Droplet Multiplexing
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Solution Overview
Problem
There is a need for improved methods of multiplexing samples and barcode tagging partitioned polynucleotides for next-generation sequencing to reduce sequencing costs while maintaining the ability to trace sequence reads back to their original samples.
Innovation Solution
The method involves partitioning organelles into droplets, lysing them to release RNA, generating tagged cDNA with barcoded adaptors, and sequencing the tagged polynucleotides to determine their origin, using techniques like transposons for tagging and polymerase chain reaction for amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of energy
If multiple samples are multiplexed for next-generation sequencing, then sequencing cost per sample is reduced, but the ability to trace sequence reads back to original samples may be compromised
Solution Approach 1:
The patent applies segmentation by dividing the sequencing process into partitioned reactions where each sample is processed in separate physical compartments (droplets or wells). This spatial segmentation allows multiple samples to be multiplexed while maintaining traceability through unique barcodes assigned to each partition, resolving the contradiction between cost reduction and information preservation.
Solution Approach 2:
The patent introduces barcodes as intermediary elements that mediate between the multiplexed samples and the sequencing reads. These unique molecular identifiers act as information carriers that link sequence data back to their original sample partitions, enabling cost-effective multiplexing without loss of sample origin information.
2Reliability
If barcodes are used to tag partitioned polynucleotides, then sample traceability is improved, but the complexity of the tagging process increases
Solution Approach 1:
The patent applies preliminary action by incorporating barcodes into the partitioning step itself, rather than adding them separately later. The barcoding occurs as part of the initial partition creation process, which simplifies the overall workflow and reduces the number of separate operations needed, thereby improving traceability without proportionally increasing complexity.
3Measurement precision
If partitions contain on average less than five organelles per partition, then single-organelle resolution is achieved, but the number of partitions required increases
Solution Approach 1:
The patent applies merging by combining multiple low-complexity partitions into a pooled sequencing reaction. After individual partitions are created with single-organelle resolution and barcoded, they are merged into a single sequencing run, which maintains precision while improving throughput by utilizing the full sequencing capacity for multiple partitions simultaneously.
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 cost-effective sequencing by enabling the pooling of samples with unique barcodes, ensuring that sequence reads can be traced back to their original partitions, thereby enhancing the efficiency and accuracy of next-generation sequencing.
Implementation Method 1
partitioning organelles into a plurality of partitions, wherein each partition comprises on average less than five organelles per partition; In some embodiments, the first partitions are droplets. In some instances, said second partitions are droplets. In some cases, said droplets are within an immiscible fluid.
Implementation Method 2
lysing said organelles in the plurality of partitions, wherein the lysing releases RNA from said organelles
Implementation Method 3
the generating tagged cDNA comprises reverse transcription of the released RNA with partition-specific barcoded primers
Implementation Method 4
Often, the amplification comprises a polymerase chain reaction
Data Source
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AI summary
Provided herein are methods, compositions, and kits for assays, many of which involve amplification reactions such as digital PCR or droplet digital PCR. The assays may be used for such applications as sequencing, copy number variation analysis, and others. In some cases, the assays involve subdividing a sample into multiple partitions (e.g., droplets) and merging the partitions with other partitions that comprise adaptors with barcodes.