Bar-coded Primer Synthesis for Multiplex Sequencing
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Solution Overview
Problem
Current DNA barcoding methods are inefficient for multiplex sequencing of large collections of samples targeting specific genomic regions, limiting their application in diagnostic purposes, particularly for personalized diagnostics and high-throughput single-cell sequencing due to high costs and time requirements.
Innovation Solution
A method for producing bar-coded primers that involve combining oligonucleotides with adapter sequences, allowing for the annealing and extension of primer sequences, enabling efficient reverse transcription and amplification of multiple nucleic acid molecules with sample-specific bar-codes, which can be used for multiplex sequencing without the need for ligation or nested PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional DNA barcoding methods are used for multiplex sequencing of large collections of samples, then sample identification can be achieved, but the process becomes costly and time-consuming, reducing productivity
Solution Approach 1:
The patent combines barcoding and primer synthesis into a single integrated process. The bar-coded primer is synthesized in one reaction step rather than requiring separate barcoding and amplification steps, thereby merging multiple operations into one and significantly increasing throughput for multiplex sequencing of hundreds to tens of thousands of samples
Solution Approach 2:
The patent incorporates the bar-code sequence directly into the primer during the synthesis process, performing the barcoding action in advance before the actual sequencing experiment. This preliminary integration of barcodes into primers eliminates the need for subsequent barcoding steps and reduces overall processing time
2Ease of manufacture
If traditional barcoding methods involving ligation or nested PCR are used, then barcoding can be achieved, but the process complexity increases and manufacturing efficiency decreases
Solution Approach 1:
The patent extracts and eliminates the complex steps of ligation and nested PCR from the barcoding process. Instead of using these multi-step methods, the invention directly synthesizes bar-coded primers in a single reaction, removing unnecessary complexity and significantly simplifying the manufacturing process
Solution Approach 2:
The patent uses a template-based synthesis approach where a template strand containing the bar-code sequence is used to create complementary bar-coded primers. This copying mechanism allows for efficient, accurate replication of the bar-code sequence without requiring complex ligation or amplification steps
3Measurement precision
If individual primer synthesis for each sample is performed, then sample-specific barcodes can be generated, but the cost and time requirements increase significantly
Solution Approach 1:
The patent creates a universal barcoding system where a single template strand can be used to generate bar-coded primers for multiple samples simultaneously. This multi-functional approach allows one template to serve many purposes, enabling parallel processing of hundreds to tens of thousands of samples while maintaining accurate sample-specific identification through unique bar-code sequences
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 enables high-throughput, cost-effective multiplex sequencing of hundreds to tens of thousands of samples, allowing for efficient barcoding and identification of individual samples, thereby facilitating targeted sequencing and gene expression analysis.
Implementation Method 1
steps (a)(i) and (a)(ii) are carried out under conditions that enable the annealing of the first and second adapter nucleic acid sequences to the respective reverse complementary sequences thereof
Implementation Method 2
extending the oligonucleotides of (a)(i) and (a)(ii) by polymerase-mediated oligonucleotide synthesis
Data Source
AI summary
The present invention relates to a method of producing a set of primers suitable for the reverse transcription and/or amplification of a plurality (N) of nucleic acid molecules of interest, wherein for each nucleic acid molecule of interest at least one primer is produced and wherein the primers carry a bar-code, the method comprising the steps of: (a)(i) combining (1) a first oligonucleotide, wherein said first oligonucleotide comprises a first bar-code nucleic acid sequence linked at its 3′ end to a first adapter nucleic acid sequence with (2) a plurality (N) of second oligonucleotides, wherein each second oligonucleotide comprises the reverse complementary sequence of a forward primer specific for a nucleic acid molecule of interest, wherein said reverse complementary sequence of the forward primer is linked at its 3′ end to the reverse complementary sequence of the first adapter nucleic acid sequence; and/or (a)(ii) combining (1) a third oligonucleotide, wherein said third oligonucleotide comprises a second bar-code nucleic acid sequence linked at its 3′ end to a second adapter nucleic acid sequence with (2) a plurality (N) of fourth oligonucleotides, wherein each fourth oligonucleotide comprises the reverse complementary sequence of a reverse primer specific for said nucleic acid molecule of interest, wherein said reverse complementary sequence of the reverse primer is linked at its 3′ end to the reverse complementary sequence of the second adapter nucleic acid sequence; wherein steps (a)(i) and (a)(ii) are carried out under conditions that enable the annealing of the first and second adapter nucleic acid sequences to the respective reverse complementary sequences thereof; (b) extending the oligonucleotides of (a)(i) and (a)(ii) by polymerase-mediated oligonucleotide synthesis; and (c) optionally, removing the second and fourth oligonucleotides. The present invention further relates to methods of producing a plurality (M) of nucleic acid amplification products of interest carrying at least one sample-specific bar-code as well as to a method for multiplex sequencing of a plurality (M) of nucleic acid amplification products of interest from a plurality (X) of samples in a single reaction chamber and identifying the individual sample from which each nucleic acid amplification product is derived. Furthermore, the present invention relates to a target-unspecific bar-code-adapter panel, its use in the methods of the invention as well as a kit comprising same.