Sequencing Direct Repeats Using Dual Outer Primers

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Solution Overview

Problem

Current sequencing methods face challenges in accurately comparing sequences with a single sequence read due to difficulties in identifying sequence beginnings and ends, especially with repeated sequences causing slippage and erroneous results, particularly in samples with limited DNA or low copy number mutations.

Innovation Solution

A method involving hybridizing primers to specific sites upstream of repeat sequences in a direct repeat template, followed by sequencing-by-synthesis to merge sequence reads from both primers, allowing for the identification of differences as low-quality base calls and exclusion of errors.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Device complexity

If a single sequence read is used to compare sequences, then the sequencing process is simplified, but alignment accuracy deteriorates due to slippage in repeated sequences

Engineering Contradiction:

Improvesequencing process complexityVSAvoidalignment accuracy

Core Design Contradiction:

Device complexityVSMeasurement precision

Solution Approach 1:

The invention divides the sequencing process into two separate sequencing reactions, each reading from a different end of the DNA fragment. This segmentation allows each read to independently locate its starting position using unique outer primer binding sites, avoiding the slippage problem in repeated sequences while maintaining manageable process complexity through systematic execution of both reactions.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces outer primers that bind to unique sequences flanking the repeated regions as intermediary elements. These outer primers serve as reliable starting points for sequencing, mediating the initiation of reads without being affected by the repeated sequences in the middle of the fragment, thus ensuring accurate alignment.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of operation

If software automatically identifies sequence beginnings and ends, then manual intervention is reduced, but reliability deteriorates due to inconsistent identification across different sequence compositions

Engineering Contradiction:

Improvemanual intervention requirementVSAvoidconsistent identification accuracy

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The invention creates local uniqueness at the ends of DNA fragments by incorporating outer primers that bind to unique sequences flanking the repeated regions. This local quality differentiation ensures that the starting and ending points of sequences are uniquely identifiable, providing reliable automatic identification by software without requiring manual intervention or dealing with inconsistencies in repeated sequences.

Inventive Principle:

Principle #3Local quality

3Adaptability or versatility

If repeated sequences are present in the template, then the template represents natural genomic structure, but measurement precision deteriorates due to alignment slippage

Engineering Contradiction:

Improvenatural genomic structure representationVSAvoidalignment accuracy

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

Instead of attempting to sequence through the repeated sequences from one end, the invention sequences from both ends simultaneously, working towards the middle. This inverted approach allows the sequencing reads to start from unique outer regions and converge on the repeated sequences, eliminating the slippage problem while still accurately representing the natural genomic structure including the repeats.

Inventive Principle:

Principle #13The other way round (Inversion)

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 effectively identifies damaged nucleotides and amplification errors, enhancing the accuracy of sequence analysis in samples with limited DNA or low copy number mutations, such as in circulating tumor DNA samples.

Implementation Method 1

hybridizing a primer to a first site that is upstream of the first repeat sequence and hybridizing a primer to a second site that is upstream of the second repeat sequence

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

sequencing the template using a sequencing-by-synthesis method (e.g., using fluorescent dye terminators) to produce a sequence read

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentEP3938541B9Method for sequencing a direct repeat

Publication Date: 2023.10.04 GENOME RES LTD

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AI summary

Described herein is a method of sequencing a template that comprises a direct repeat, comprising: (a) in the same reaction, hybridizing a primer to a first site that is upstream of the first repeat sequence and hybridizing a primer to a second site that is upstream of the second repeat sequence, wherein the first and second sites are: (i) upstream of the first and second repeat sequences, respectively, and (ii) equidistant from the first and second repeat sequences; and (b) subjecting the hybridization product of (a) to a sequencing-by-synthesis sequencing reaction to produce a sequence read that comprises a combination of the first and second repeat sequences.