TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting.
Hindrek TederMariann KoelPriit PaluojaTatjana JatsenkoKadri RekkerTriin Laisk-PodarViktorija KukuškinaAgne Velthut-MeikasOlga FjodorovaMaire PetersJuha KereAndres SalumetsPriit PaltaKaarel KrjutškovPublished in: NPJ genomic medicine (2018)
Targeted next-generation sequencing (NGS) methods have become essential in medical research and diagnostics. In addition to NGS sensitivity and high-throughput capacity, precise biomolecule counting based on unique molecular identifier (UMI) has potential to increase biomolecule detection accuracy. Although UMIs are widely used in basic research its introduction to clinical assays is still in progress. Here, we present a robust and cost-effective TAC-seq (Targeted Allele Counting by sequencing) method that uses UMIs to estimate the original molecule counts of mRNAs, microRNAs, and cell-free DNA. We applied TAC-seq in three different clinical applications and compared the results with standard NGS. RNA samples extracted from human endometrial biopsies were analyzed using previously described 57 mRNA-based receptivity biomarkers and 49 selected microRNAs at different expression levels. Cell-free DNA aneuploidy testing was based on cell line (47,XX, +21) genomic DNA. TAC-seq mRNA profiling showed identical clustering results to transcriptome RNA sequencing, and microRNA detection demonstrated significant reduction in amplification bias, allowing to determine minor expression changes between different samples that remained undetermined by standard NGS. The mimicking experiment for cell-free DNA fetal aneuploidy analysis showed that TAC-seq can be applied to count highly fragmented DNA, detecting significant (p = 7.6 × 10-4) excess of chromosome 21 molecules at 10% fetal fraction level. Based on three proof-of-principle applications we demonstrate that TAC-seq is an accurate and highly potential biomarker profiling method for advanced medical research and diagnostics.
Keyphrases
- single cell
- rna seq
- high throughput
- circulating tumor
- nucleic acid
- single molecule
- poor prognosis
- cell free
- healthcare
- binding protein
- copy number
- endothelial cells
- genome wide
- loop mediated isothermal amplification
- peripheral blood
- high resolution
- dna methylation
- drug delivery
- long non coding rna
- circulating tumor cells
- quantum dots
- pluripotent stem cells