Nucleic Acid Adaptor Labelling With In Situ Random Barcodes
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Solution Overview
Problem
Current nucleic acid sequencing methods face challenges in detecting genetic alterations below 1% allele frequency due to errors introduced during processing, leading to difficulties in distinguishing true variants from processing errors, and existing molecular barcoding techniques are costly, complex, and inefficient.
Innovation Solution
In situ generation of molecular barcodes using adaptors with universal nucleotide bases that allow random tag incorporation through an extension reaction, eliminating the need for pre-synthesized complex barcode mixes and reducing error propagation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If complex molecular barcodes with high diversity are synthesized and attached to NGS libraries, then the ability to detect genetic alterations at low allele frequency is improved, but the cost and complexity of the process increases
Solution Approach 1:
The patent incorporates barcodes during the PCR amplification step itself, rather than requiring separate pre-synthesis and pooling steps. The barcode is built up as the DNA polymerase extends the primer, integrating the tagging function into the existing amplification workflow.
Solution Approach 2:
The patent combines the barcode synthesis and library tagging steps into a single PCR reaction. The barcode-generating primer and template DNA are amplified simultaneously, merging multiple functions (amplification, tagging, and library preparation) into one process.
2Reliability
If a high degree of tag diversity is required for efficient sequencing, then the ability to distinguish true variants from errors is improved, but the cost and time required for separate barcode synthesis reactions increases
Solution Approach 1:
The barcode diversity is generated in advance during the PCR amplification process itself. By the time sequencing occurs, the full diversity of barcodes is already present on the amplified molecules, eliminating the need for time-consuming post-PCR barcode pooling steps.
Solution Approach 2:
The system generates its own barcode diversity internally during the PCR reaction, rather than requiring external synthesis and pooling operations. The random incorporation of nucleotides during amplification naturally creates the diverse barcode population needed for reliable variant detection.
3Ease of manufacture
If molecular barcodes are synthesized as single-stranded oligonucleotides and attached by PCR, ligation or primer extension, then the tagging function is achieved, but error propagation during processing occurs
Solution Approach 1:
The barcode is divided into two functional segments: a constant portion that provides the unique molecular identifier and a variable portion that is randomly generated during PCR. This segmentation allows the stable identification function to be separated from the error-prone random generation process.
Solution Approach 2:
The constant barcode sequence is copied faithfully during PCR amplification, while the random portion is generated de novo each cycle. This copying mechanism ensures that the identification function is preserved accurately across all amplifications, reducing error propagation.
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 specificity and sensitivity in detecting genetic alterations, reduces errors, and simplifies the tagging process while maintaining high tag diversity, allowing for accurate differentiation between true variants and processing errors.
Implementation Method 1
utilising adaptors having universal nucleotide bases that allow the incorporation of randomly generated tags in a nucleotide extension reaction
Data Source
AI summary
The invention relates to methods for labelling individual nucleic acid molecules present in a sample, comprising contacting the nucleic acid molecules with an adaptor or mixture of adaptors, wherein the adaptor or adaptors comprise one or more universal nucleotide bases and a ligation moiety at their 3′ end, and ligating an adaptor to the nucleic acid of interest, wherein the adaptor is ligated to the nucleic acid molecules at the 3′ end of the adaptor. A random tag is then generated in situ by conducting an extension reaction over the ligated adaptor. Methods of the invention may be used to detect genetic alterations or variants in any nucleic acid with high specificity and high sensitivity, including mutations in nucleic acids such as ctDNA, cfDNA, and in viral, microbiome and plant nucleic acids. Methods of the invention may also be used in detection and correction of errors introduced into nucleic acids during processing.