Solid-Substrate Nucleic Acid Barcoding for Low-Input Sequencing
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Solution Overview
Problem
Existing methods for nucleic acid sequencing, particularly in low DNA input scenarios, are inefficient and labor-intensive, with high risks of contamination and error, especially in applications like preimplantation genetic testing.
Innovation Solution
A method involving clonal amplification on a solid substrate using degenerate and fixed nucleotide sequences, combined with adapter sequences for priming, to produce barcoded nucleic acids for sequencing, reducing hands-on time and reagent use.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If whole genome amplification is performed before sequencing, then sufficient DNA for library preparation is obtained, but operator hands-on time and total protocol time increase
Solution Approach 1:
The patent combines whole genome amplification and PCR barcoding into a single simultaneous reaction rather than sequential steps. The amplifying primer contains both the degenerate sequence for WGA and the specific identifier sequence for barcoding, allowing both functions to occur in one protocol, thereby reducing operator hands-on time and total protocol time while still producing sufficient amplified DNA for sequencing.
Solution Approach 2:
The amplifying primer serves multiple functions: it acts as a degenerate primer for whole genome amplification, incorporates a specific identifier sequence for barcoding, and includes a 5' fixed sequence for adapter annealing. This multi-functional primer design eliminates the need for separate WGA and barcoding steps, streamlining the workflow.
2Quantity of substance
If sequential WGA followed by standard NGS library preparation is used, then DNA amplification is achieved, but reagent requirements and protocol complexity increase
Solution Approach 1:
The patent merges the WGA and barcoding steps into a single simultaneous reaction. The amplifying primer integrates the degenerate sequence for genome-wide amplification with the specific identifier sequence for sample identification, eliminating the need for separate sequential operations and reducing protocol complexity.
Solution Approach 2:
The specific identifier sequence (barcode) is incorporated into the amplifying primer during the initial WGA step, so that barcoding occurs as a preliminary action within the amplification process itself, rather than requiring a subsequent separate barcoding step before library preparation.
3Loss of information
If standard PCR barcoding is performed after WGA, then sample identification is achieved, but additional reagents and hands-on time are required
Solution Approach 1:
The amplifying primer is designed with multiple functional elements: a degenerate sequence for WGA, a specific identifier sequence for barcoding, and a 5' fixed sequence for adapter annealing. This single multi-functional primer replaces what would traditionally require separate primers and reagents for WGA and barcoding, reducing overall reagent requirements.
Solution Approach 2:
The patent combines the barcoding function with the amplification function in a single simultaneous reaction. The specific identifier sequence is incorporated during the WGA process itself, eliminating the need for a separate post-WGA barcoding step and its associated reagents and hands-on time.
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
Enhances laboratory efficiency by decreasing operator error, reducing contamination risk, and streamlining workflows while maintaining sequencing accuracy, particularly suitable for low DNA input samples.
Implementation Method 1
amplifying the nucleic acid sample using an amplifying primer comprising a degenerate nucleotide sequence and a 5′ fixed nucleotide sequence
Implementation Method 2
amplifying the nucleic acid sample using an amplifying primer... to produce amplified nucleic acids
Implementation Method 3
the first adapter nucleotide sequence or the second adapter nucleotide sequence provides a sequence for subsequent priming of DNA synthesis from a nucleotide sequence attached to the solid substrate
Data Source
AI summary
The present disclosure relates to methods, kits and products for barcoding of nucleic acids. In certain embodiments, the present disclosure provides a method of producing nucleic acid for sequencing utilising clonal amplification on a solid substrate, the method comprising: (a) providing a nucleic acid sample for sequencing; (b) amplifying the nucleic acid sample using an amplifying primer comprising a degenerate nucleotide sequence and a 5′ fixed nucleotide sequence, to produce amplified nucleic acids; and (c) further amplifying the amplified nucleic acids with (i) a first primer comprising the 5′ fixed nucleotide sequence and a first adapter nucleotide sequence, and (ii) a second primer comprising the 5′ fixed nucleotide sequence and a second adapter nucleotide sequence, wherein the first adapter nucleotide sequence or the second adapter nucleotide sequence provides a sequence for subsequent priming of DNA synthesis from a nucleotide sequence attached to the solid substrate and the other adapter nucleotide sequence provides a sequence for subsequent priming of DNA synthesis from a template produced from the subsequent priming, and wherein one or more of the first primer, the second primer and the amplifying primer comprise a specific identifier sequence to identify nucleic acids amplified with the first primer, the second primer and/or the amplifying primer; thereby producing nucleic acid for sequencing utilising clonal amplification on the solid substrate.