Abstract
Whole-molecule reconstruction from short reads requires more than sequencing depth: the fragments must retain a reliable connection to the molecule from which they originated. PSeq addresses that requirement by introducing a multifunctional tag during reverse transcription and redistributing its marker among random fragments generated from the tagged cDNA. The marker carries a high-diversity Source Molecule Identifier (SMID), fixed sequence landmarks, and strand-sense information that can be recognized after paired-end sequencing.
This paper describes the molecular design and expected read structure of the PSeq library. It is an architectural description, not a reagent recipe or a validation report. Detailed kit compositions and operational procedures remain in controlled SOPs; technical evidence for validation is consolidated in Paper 4 of this series.
Key topics
- High-diversity SMID barcodes, wrappers, and invariant check bases
- Strand-sense discriminators and machine-recognizable marker design
- Intramolecular fragmentation and marker transfer
- Paired-end read structure and the chemistry-to-pipeline interface
Contents
- The identity-preservation problem
- Anatomy of the PSeq tag
- Source Molecule Identifier, wrapper, check-base, and strand-sense design
- From RNA tagging to a paired-end PSeq DNA library
- Expected sequencing-read contract
- Design variants, pipeline interfaces, and controlled SOP boundaries