Template Switch Oligonucleotide for Balanced Illumina Sequencing
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Solution Overview
Problem
Current sequencing methods using the 'Capture and Amplification by Tailing and Switching' (CATS) technology face limitations on Illumina platforms due to the template switch motif being highly enriched in Guanosine, leading to unbalanced base distribution and reduced sequencing output and quality, especially in the initial sequencing cycles.
Innovation Solution
A new Template Switching Oligonucleotide (TSO) construct is designed with a primer sequence, a template switch motif, and a random linker sequence to improve library preparation, ensuring a more balanced base distribution and facilitating successful template generation on Illumina platforms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the template switch motif is highly enriched in Guanosine to enable template switching, then template switching efficiency is improved, but base distribution balance deteriorates and sequencing output quality worsens
Solution Approach 1:
The template switch oligo is divided into distinct functional segments: a template switching motif segment (enriched in Guanosine for efficient template switching) and a random linker segment (providing balanced base distribution). This segmentation allows each segment to optimize its specific function without compromising the other, resolving the contradiction between template switching efficiency and base distribution balance.
Solution Approach 2:
Different regions of the template switch oligo are assigned different compositional qualities: the template switching motif region is locally optimized with high Guanosine content for efficient switching, while the random linker region is locally optimized with balanced base composition for improved sequencing output. This local quality differentiation resolves the contradiction by allowing specialized optimization in each region.
2Adaptability or versatility
If the template switch motif is highly enriched in Guanosine to facilitate template switching, then template switching capability is improved, but sequencing output and quality deteriorate
Solution Approach 1:
The template switch oligo is segmented into a template switching motif segment (providing adaptability for template switching) and a random linker segment (providing balanced base distribution for improved sequencing output). This segmentation resolves the contradiction between template switching capability and sequencing productivity.
Solution Approach 2:
The base composition parameters of the template switch oligo are changed in different regions: the template switching motif has high Guanosine content (parameter optimization for switching capability), while the random linker has balanced base composition (parameter optimization for sequencing output). This parameter differentiation resolves the contradiction between adaptability and productivity.
3Ease of manufacture
If a simple template switch oligo is used to minimize complexity, then ease of manufacture is improved, but sequencing performance deteriorates
Solution Approach 1:
The template switch oligo is segmented into two simple functional parts: a template switching motif and a random linker. This segmentation maintains manufacturing simplicity while improving sequencing performance through the strategic combination of these two segments with different compositional characteristics.
Solution Approach 2:
The template switch oligo is constructed as a composite of two functional segments with different base compositions: the template switching motif segment and the random linker segment. This composite structure combines the advantages of both segments, maintaining ease of manufacture while achieving improved sequencing performance.
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
The improved TSO construct enhances sequencing output and quality by achieving a more diverse nucleic acid detection, increasing positive reads, and improving sensitivity and reproducibility, especially for sensitive and degraded RNA sequences.
Implementation Method 1
hybridizing a priming oligonucleotide sequence complementary to the added nucleotide sequence and synthesizing a cDNA sequence with a template dependent DNA polymerase
Implementation Method 2
when the applied method is performed upon the MGI platform with MGI tools to obtain nanoballs, possibly adding a splint oligo sequence that hybridizes to adapter DNA sequences
Implementation Method 3
synthesizing a cDNA sequence with a template dependent DNA polymerase to obtain a double stranded nucleic acid sequence
Implementation Method 4
adding at least 5, preferably at least 10, at least 15, at least 20, at least 25, at least 30, but preferably less than 100, less than 75, less than 50 consecutive nucleotides to the 3′ terminus of the native single stranded or native double stranded nucleic acid sequence
Data Source
AI summary
The present invention is related to a Template Switch Oligo construct and its use into a ligase free diagnostic and/or sequencing method and to the kit for performing the method of the invention.