TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Targeted next-generation sequencing (NGS) is crucial for medical research and diagnostics.
• Unique Molecular Identifiers (UMIs) enhance biomolecule detection accuracy but are underutilized in clinical assays.
• There is a need for precise and cost-effective methods for quantifying nucleic acids in clinical settings.

Purpose of the Study:

• To introduce and validate TAC-seq, a novel UMI-based method for accurate quantification of mRNA, microRNA, and cell-free DNA.
• To assess the performance of TAC-seq in clinical applications and compare it with standard NGS methods.
• To demonstrate the potential of TAC-seq as a biomarker profiling tool for advanced diagnostics.

Main Methods:

• Developed TAC-seq, a cost-effective method utilizing UMIs for precise biomolecule counting.
• Applied TAC-seq to analyze mRNA biomarkers and microRNAs from human endometrial biopsies.
• Used TAC-seq for cell-free DNA aneuploidy testing, including fetal aneuploidy analysis with fragmented DNA.

Main Results:

• TAC-seq mRNA profiling produced clustering results identical to transcriptome RNA sequencing.
• MicroRNA detection with TAC-seq showed reduced amplification bias, enabling detection of minor expression changes missed by standard NGS.
• TAC-seq successfully detected a significant excess of chromosome 21 molecules in a cell-free DNA aneuploidy test at a 10% fetal fraction.

Conclusions:

• TAC-seq is a robust, cost-effective method for accurate quantification of various nucleic acid types.
• The method demonstrates significant advantages over standard NGS, particularly in detecting subtle expression changes and analyzing fragmented DNA.
• TAC-seq shows high potential as a biomarker profiling tool for advanced medical research and clinical diagnostics.