%0 Journal Article
%T TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting
%A Agne Velthut-Meikas
%A Andres Salumets
%A Hindrek Teder
%A Juha Kere
%A Kaarel Krjut£¿kov
%A Kadri Rekker
%A Maire Peters
%A Mariann Koel
%A Olga Fjodorova
%A Priit Palta
%A Priit Paluoja
%A Tatjana Jatsenko
%A Triin Laisk-Podar
%A Viktorija Kuku£¿kina
%J Archive of "NPJ Genomic Medicine".
%D 2018
%R 10.1038/s41525-018-0072-5
%X Principle and technical parameters of TAC-seq. a Schematic diagram of the assay to detect specific mRNA or cell-free DNA. Target-specific DNA oligonucleotide detector probes hybridize under stringent conditions to the studied cDNA or cfDNA. Both detector oligonucleotides consist of a specific 27-bp region (green), 4-bp unique molecular identifier (UMI) motif (NNNN), and universal sequences (purple and orange). The right detector oligonucleotide is 5¡ä phosphorylated. After rigorous hybridization, the pair of detector probes is ligated using a thermostable ligase under stringent conditions. Next, the ligated detectors complexed with the target region are captured with magnetic beads and PCR amplified to introduce sample-specific barcodes and other common motifs that are required for single-read NGS. b Spearman correlation analysis of the input and detected ERCC synthetic spike-in mRNA molecules at UMI threshold 4 (UMI£¿=£¿4). UMI threshold is defined as the number of detected unique UMI sequences. For example, UMI£¿=£¿4 indicates that a certain UMI motif is detected at least four times. UMIs are valuable only if the number of UMI combinations (8-bp UMI provides 65,536 variants, for example) is substantially larger than the sum of the target molecules in the studied sample. c Bar plot of Spearman¡¯s correlation analysis of the ERCC input and detected molecules at different UMI thresholds. d Reproducibility of seven technical ERCC replicates (seven different icons on plot) of 22 spike-in molecules at UMI£¿=£¿
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299075/