ESPeR-seq: Extremely Sensitive and Pure, End-to-end, RNA-seq library preparation.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Smart-seq methods are standard for single-cell RNA sequencing but face challenges with PCR bias, imprecise transcription end site (TES) capture, and phantom UMIs.
• Phantom UMIs inflate molecular counts, compromising RNA sequencing data accuracy.
• Accurate quantification and full-length transcript resolution are crucial for single-cell analysis.

Purpose of the Study:

• To introduce ESPeR-seq, a novel single-cell RNA sequencing architecture designed to address existing limitations.
• To enable precise, stranded transcription end site (TES) capture.
• To eliminate PCR background and phantom UMIs for improved molecular count accuracy.

Main Methods:

• Developed an "Omega-dT" primer for direct, high-quality sequencing at transcript termini and precise, stranded TES capture.
• Implemented a biochemical "multi-lock" mechanism using uracil-containing TSOs and a uracil-intolerant DNA polymerase to prevent PCR bias and phantom UMIs.
• Introduced the logQ-slope metric for sensitive diagnosis of UMI fidelity.

Main Results:

• ESPER-seq strictly prevents UMI inflation, unlike current state-of-the-art methods.
• The method achieves precise, stranded TES capture, facilitating robust de novo gene model reconstruction.
• Discovered novel multi-exon genes, unannotated 3' UTR extensions, and candidate eRNAs.

Conclusions:

• ESPER-seq provides a robust framework for absolute quantitative accuracy in single-cell RNA sequencing.
• The technology enables high-resolution, full-length isoform analysis.
• ESPER-seq significantly advances the capabilities of single-cell transcriptomics for gene discovery and quantification.