Barcoded DNA Constructs for Accurate Short-Fragment Nanopore Sequencing

The invention segments the DNA sequencing process by dividing long DNA fragments into multiple short sub-fragments that can be individually barcoded and sequenced. Each sub-fragment is assigned a unique molecular identifier (UMI) and barcode, allowing the original long fragment to be reconstructed from multiple accurate short-read sequences, thereby resolving the contradiction between handling long fragments and maintaining accuracy.

The invention introduces barcodes and UMIs as intermediary elements that mediate between the long DNA fragment and the sequencing process. These intermediaries enable tracking and grouping of sub-fragments, allowing accurate reconstruction of the original sequence by comparing multiple reads of the same sub-fragment, thus improving accuracy while maintaining the ability to handle long fragments.

The invention applies preliminary action by performing PCR amplification with high-fidelity polymerases and incorporating barcodes/UMIs during the amplification step itself. This allows error correction to be built into the amplification process, where multiple copies with identical barcodes can be consensus-called to correct polymerase errors, thus achieving both sufficient DNA quantity and high accuracy.

The invention implements feedback mechanisms by using barcodes to group PCR replicates and UMIs to track individual molecular copies. This enables computational consensus calling where sequencing errors or PCR errors are identified and corrected by comparing multiple reads of the same barcoded sub-fragment, providing feedback-based error correction that maintains accuracy while amplifying DNA quantity.

The invention adds another dimension to the sequencing approach by organizing short DNA fragments into barcoded groups that are ligated to form longer composite structures suitable for nanopore sequencing. This dimensional transformation allows short fragments to be processed through a long-fragment-optimized platform by embedding them in a larger contextual framework that the platform can handle efficiently.

The invention creates a universal sequencing construct that can accommodate both short and long DNA fragments. The barcoded sub-fragment design with introducer and closure sequences provides a universal framework that works regardless of the original fragment length, allowing the nanopore platform to efficiently sequence various fragment types through a unified process, thus improving adaptability.