Article Title: TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting
Figure Lengend Snippet: 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’ 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=4.
Article Snippet: After ligation, the free ligation adapter was removed by adding 0.5 µl 5’-Deadenylase (25 U/µl, NEB) and 0.5 µl Lambda exonuclease (5 U/µl, NEB) and incubated 10 min at 37°C, followed by 10 min at 75°C. cDNA was synthesized after adding 0.4 µl 100 mM DTT (Invitrogen), 0.4 µl 2 M KCl (Sigma-Aldrich), 0.4 µl 10 mM dNTPs (Thermo Fisher), 0.4 µl RNase inhibitor (Thermo Fisher), 0.2 µl 10 µM micro RT biotin primer and 0.2 µl Maxima H Minus Reverse Transcriptase (200 U/µl, Thermo Fisher) which were mixed into one 2 µl master mix. cDNA incubation was carried out for 15 min at 50°C, followed by 5 min at 80°C.
Techniques: Hybridization, Magnetic Beads, Polymerase Chain Reaction, Amplification, Introduce, Next-Generation Sequencing