Multiplexed Sequencing Primers for Overlapping Cluster Reads
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
RNA sequencing experiments typically sequence short reads (25 to 75 base pairs) inefficiently, underutilizing sequencing space and overusing reagents and resources on commercial platforms designed for longer reads.
Innovation Solution
A method involving multiple sequencing primers and modified nucleotides with reversible terminators for sequencing different populations of polynucleotides, allowing for sequential incorporation of complementary nucleotides and generating multiple sequencing reads, and amplifying template polynucleotides to form overlapping clusters on a solid support.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If short read sequencing (25-75 base pairs) is performed on commercial platforms designed for longer reads, then sequencing experiments can be completed, but sequencing space is underutilized and reagent consumption is excessive
Solution Approach 1:
The flow cell surface is divided into multiple discrete regions, each containing a specific pool of sequencing primers. Different template populations are selectively sequenced in different regions, allowing efficient utilization of sequencing space while reducing unnecessary reagent consumption across the entire flow cell.
Solution Approach 2:
Different regions of the flow cell are assigned different primer pools with specific sequences, creating local variations in sequencing capability. This allows each region to be optimized for specific template populations, improving overall sequencing efficiency and reducing waste of universal reagents.
2Productivity
If multiple different sequencing primers are used to sequence different template populations, then sequencing specificity and efficiency improve, but system complexity increases
Solution Approach 1:
A single flow cell surface is designed to support multiple primer pools, each with different specificities. The system can selectively activate different primer pools for different template populations, achieving multi-functionality without requiring separate sequencing systems for each template type.
Solution Approach 2:
The complexity of managing multiple primers is resolved by adding a spatial dimension - primers are physically separated into different regions on the flow cell surface. This spatial organization simplifies primer pool management and allows parallel sequencing of multiple template populations without cross-contamination.
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 sequencing efficiency by utilizing sequencing space more effectively and reducing reagent consumption, enabling longer read lengths and improved data output.
Implementation Method 1
contacting a first population of polynucleotides annealed to a first sequencing primer with a first sequencing solution
Implementation Method 2
monitoring the sequential incorporation of complementary nucleotides to generate a first sequencing read, wherein the reversible terminator is removed prior to the introduction of the next complementary nucleotide
Data Source
AI summary
Disclosed herein, inter alia, are methods and compositions for sequencing a plurality of template nucleic acids. In an aspect is provided a method of sequencing an overlapping amplification cluster, a method of sequencing different polynucleotide immobilized on a solid support, and a method of sequencing three or more populations of polynucleotides on a solid support.