Phosphorothioate Oligonucleotide Adapters for FFPE Sequencing
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Solution Overview
Problem
Current methods for studying mutational signatures in cancer biology, such as Whole Genome Sequencing (WGS), are limited by high costs and inability to analyze low-quality or formalin-fixed paraffin-embedded (FFPE) samples, which are common in clinical settings.
Innovation Solution
A novel approach involving modified oligonucleotide adapters and a quasi-random sampling method using restriction enzymes to generate sequencing libraries, allowing for efficient ligation and enzymatic clean-up, enabling analysis of FFPE samples with improved data quality comparable to WGS at a lower cost.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Whole Genome Sequencing (WGS) is used to study mutational signatures, then data quality and comprehensiveness are improved, but cost and applicability to low-quality/FFPE samples worsen
Solution Approach 1:
The patent applies segmentation by using restriction enzymes to divide the genome into discrete fragments before sequencing. This reduces the sequencing scope from whole genome to targeted fragments, lowering cost and improving feasibility for FFPE samples while maintaining data quality through careful fragment selection and assembly.
Solution Approach 2:
The patent implements local quality by focusing sequencing efforts on specific genomic regions identified through restriction enzyme digestion patterns. Instead of uniform whole-genome coverage, the method prioritizes regions with characteristic restriction sites, optimizing resource allocation for the most informative areas while reducing overall cost.
2Ease of manufacture
If restriction enzyme-based reduced representation sequencing is used, then cost and sample quality requirements are improved, but sequencing coverage and completeness worsen
Solution Approach 1:
The patent applies preliminary action by performing restriction enzyme digestion and fragment preparation before sequencing. This pre-processing step enriches for informative fragments with restriction sites, ensuring that the subsequent sequencing focuses on high-value regions while maintaining cost-effectiveness and sample compatibility.
Solution Approach 2:
The patent uses copying by creating adapter-ligated copies of restricted DNA fragments that can be amplified and sequenced. The adapters serve as templates for PCR amplification, generating multiple copies of each fragment for sequencing, thereby improving coverage without requiring whole-genome sequencing.
3Ease of operation
If standard oligonucleotide adapters are used for library construction, then ease of ligation is improved, but adapter dimer formation and ligation efficiency worsen
Solution Approach 1:
The patent applies local quality to adapter design by incorporating phosphorothioate modifications at specific locations within the adapter sequence. These localized chemical modifications provide nuclease resistance and prevent adapter dimer formation at critical positions, improving ligation efficiency without compromising overall ligation ease.
Solution Approach 2:
The patent uses composite materials by combining standard adapter sequences with phosphorothioate chemical modifications. This hybrid structure merges the ease of standard adapter ligation with the enhanced stability and reduced dimerization properties of chemically modified adapters, achieving both ligation ease and high productivity.
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 method provides cost-effective, high-quality sequencing data from problematic sample types like FFPE, overcoming the limitations of existing technologies and enabling universal applicability across various tumor types with reduced representation of the genome.
Implementation Method 1
at least the 6 bases at the 3' terminal end of the bottom strand are each phosphorothioated
Implementation Method 2
contacting said DNA with a pair of oligonucleotide adapters and at least one DNA ligase; and incubating to allow annealing of the oligonucleotide adapters to the digested DNA and ligation of the annealed oligonucleotide adapters to the digested DNA
Implementation Method 3
contacting said DNA with a first restriction enzyme and a second restriction enzyme; and incubating to allow digestion of the DNA by the first restriction enzyme and the second restriction enzyme
Data Source
AI summary
The invention relates to a method of preparing a nucleic acid library from a sample comprising high molecular weight DNA (HMW DNA), preferably genomic DNA, comprising the steps (i) contacting said DNA with a first restriction enzyme and a second restriction enzyme; (ii) contacting said DNA with a pair of oligonucleotide adapters according to any of claims 1 to 12; (iii) contacting said DNA with at least one DNA ligase; and (iv) incubating to allow digestion of the DNA by said first restriction enzyme and second restriction enzyme, annealing of said oligonucleotide adapters to the digested DNA, and ligation of the annealed oligonucleotide adapters to the digested DNA by said at least one DNA ligase. The invention also relates to oligonucleotide adapters, a kit, and uses of same.