Circularizing Long DNA Ends for High-Throughput Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for sequencing the ends of long linear DNAs, such as those in phage-displayed antibody libraries, are limited by low throughput, making it challenging to assess the diversity of both heavy chain and light chain variable regions simultaneously.
Innovation Solution
A method involving the fusion of 5' and 3' end segments of linear double-stranded DNA molecules, separated by a spacer, to form a circular molecule, which is then sequenced using high-throughput techniques, allowing for the simultaneous sequencing of both ends.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Sanger sequencing is used to sequence both heavy chain and light chain variable regions, then sequence accuracy is maintained, but throughput is limited and only a small number of clones can be assessed
Solution Approach 1:
The DNA molecule is segmented into distinct regions: the 5' end segment containing the heavy chain variable region, the 3' end segment containing the light chain variable region, and a spacer segment separating them. This segmentation allows each region to be optimized for its specific sequencing requirements while maintaining overall structural integrity for high-throughput processing.
Solution Approach 2:
A spacer segment is introduced as an intermediary element between the 5' and 3' end segments. This spacer acts as a mediator that enables the two ends to be brought together in a configuration suitable for high-throughput sequencing while maintaining the separation and identity of the heavy chain and light chain variable regions.
2Productivity
If high throughput parallel sequencing is used to read a vast population of the library, then productivity increases, but it remains challenging to sequence both heavy chain and light chain variable regions at the same time
Solution Approach 1:
The 5' end segment and 3' end segment are merged into a single circularized DNA molecule through ligation. This merging allows both segments to be sequenced simultaneously in a single high-throughput sequencing reaction, eliminating the need for separate sequencing operations and reducing overall complexity while maintaining high productivity.
Solution Approach 2:
The linear DNA structure is transformed into a circular configuration by joining the 5' and 3' ends. This dimensional change from linear to circular topology enables the sequencing machine to read both ends of the original linear molecule in a single continuous read, simplifying the sequencing process while increasing throughput.
3Productivity
If the 5' and 3' end segments are joined to form a circular molecule for high throughput sequencing, then both ends can be sequenced simultaneously, but additional steps such as ligation and circularization are required
Solution Approach 1:
The 5' and 3' end segments are prepared with compatible ends in advance, allowing them to be joined together in a single ligation step. This preliminary preparation simplifies the overall process by reducing the number of separate operations needed and making the circularization step more straightforward, thereby maintaining ease of manufacture while achieving high throughput.
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
Enables high-throughput sequencing of both heavy chain and light chain variable regions, significantly improving the assessment of antibody library diversity and speeding up the screening process for antigen-specific enrichment.
Implementation Method 1
incubating the dsDNA molecule under conditions permitting annealing of the two ends via a sequence of the adaptor
Implementation Method 2
a ligase to seal a nick
Data Source
AI summary
This invention relates to linking, amplifying and sequencing of two ends of long linear DNAs. In particular, this invention provides methods for pairing and sequencing VH and VL genes that encode two parts of one immunoglobulin. The method of the present invention can be applied to rapid antibody discovery and engineering.