GSM7431044: RIV A8 803 bp C template 2 µM dGTP and 200 µM d(A/T/C)TP (MiSeq); synthetic construct; OTHER
Reaction mixtures for linear PCR contained 2 µM dGTP and 200 µM d(A/T/C)TP (each), 100 nM 109 bp forward primer, 50 pM 803 bp C or 5mC template and 250 nM KTq variant in 1x KTq reaction buffer (50 mM Tris HCl (pH 9.2), 16 mM (NH4)2SO4, 2.5 mM MgCl2, 0.1% (v/v) Tween 20). PCR was performed in 25 µL with an initial denaturation at 95°C for 3 min followed by amplification over 20 cycles with denaturation at 95°C for 10 s, annealing at 62°C for 30 s and elongation at 72°C for 4 min. Final elongation was performed for 10 min at 72°C. Single-stranded (ssDNA) PCR product from 109 to 364 nt was purified by preparative agarose gel electrophoresis using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel) in combination with the NTC binding buffer according to the manufacturer's instructions with elution in 22 µL Milli-Q water. To verify that no original template DNA was extracted, 1 µL of purified PCR product was subsequently applied in a PCR for purity verification. Reaction mixtures contained 200 µM dNTPs (each), 500 nM 803 bp forward and reverse primer, 10% (v/v) PCR product as template and 0.02 U/µL Q5® Hot Start High-Fidelity DNA Polymerase in 1x Q5® Reaction Buffer (New England Biolabs). PCR was performed in 10 µL reaction mixtures with an initial denaturation at 98°C for 1 min followed by amplification over 25 cycles with denaturation at 98°C for 10 s, annealing at 62°C for 30 s and elongation at 72°C for 30 s. Final elongation was performed for 2 min at 72°C. The absence of the 803 bp PCR product was verified by agarose gel electrophoresis. 17.5 µL of purified linear PCR product ssDNA was treated with the PreCR® Repair Mix (New England Biolabs) in 20 µL according to the manufacturer's instructions. Without further purification, the reaction mixture was applied in a 2-cycle UMI PCR. Reaction mixtures for UMI introduction contained 200 µM dNTPs (each), 200 nM forward and reverse UMI primer, 0.02 U/µL Q5® Hot Start High-Fidelity DNA Polymerase and 0.2x Q5® Reaction Buffer (New England Biolabs) in 25 µL end volume. PCR was performed with an initial denaturation at 98°C for 1 min followed by primer elongation over 2 cycles with denaturation at 98°C for 10 s, annealing at 48°C for 30 s and elongation at 72°C for 30 s. Final elongation was performed for 2 min at 72°C. 183 bp UMI PCR product was purified by preparative agarose gel electrophoresis using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel) according to the manufacturer's instructions. To improve purification, NTI binding buffer was 1:1.5 diluted with Milli-Q water for solubilising agarose gel slices and the dissolved mixture was diluted in total to 1:2.5 prior to DNA binding on the purification column. Purified UMI PCR product was eluted in 15 µL Milli-Q water. DNA concentration was determined by qPCR using previously prepared 183 bp reference DNA in decadic dilution series for the quantification based on standard amplification curves. Reaction mixtures contained 0.96x NEBNext® Ultra™ II Q5® Master Mix (New England Biolabs), 400 nM 183 bp forward and reverse primer, 1x SYBR Green I (Sigma-Aldrich) and 1.85 µL reference template or UMI PCR product elution in 5 µL end volume. qPCR was performed in a Light Cycler® 96 instrument (Roche Diagnostics) with an initial denaturation at 98°C for 1 min followed by amplification over 30 cycles with denaturation at 98°C for 10 s, annealing at 60°C for 30 s and elongation at 72°C for 30 s. High resolution melting curves were measured immediately after PCR amplification. For absolute quantification, Cq values of reactions with reference template were plotted against the logarithm of their DNA concentrations and the linear regression function was used to calculate the concentration of the UMI PCR product. Calculated concentrations were multiplied by two, because applied reference template DNA was double stranded and only one strand of UMI PCR product carried both primer binding sites. Reactions for reference and UMI DNA were performed twice and a no template control reaction was performed to determine side product signals. The minimum reaction efficiency for the standard curve was 0.9 to 1.1 and the cut-off for the correlation coefficient of linear regression was 0.98. 67.63 fM of purified UMI PCR product was treated with the PreCR® Repair Mix (New England Biolabs) in 11.25 µL according to the manufacturer's instructions. Without further purification, the reaction mixture was applied in the Amplicon PCR. Reaction mixtures for Amplicon library preparation contained 1x NEBNext® Ultra™ II Q5® Master Mix (New England Biolabs), 400 nM forward and reverse Amplicon primer (containing Illumina TruSeq adapter and indexes sequences) and 26.57 fM UMI PCR product (for final analysis of approximately 100 000 UMI families) in 25 µL end volume. PCR was performed with an initial denaturation at 98°C for 1 min followed by amplification over 35 cycles with denaturation at 98°C for 10 s, annealing at 70°C for 30 s and elongation at 72°C for 30 s. Final elongation was performed for 2 min at 72°C. 263 bp Amplicon PCR product was purified by preparative agarose gel electrophoresis using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel) according to the manufacturer's instructions. To improve purification, NTI binding buffer was 1:1.5 diluted with Milli-Q water for solubilising agarose gel slices and the solved mixture was diluted in total to 1:6.5 prior to DNA binding on the purification column. The PCR product was eluted in 20 µL Milli-Q water and treated with the PreCR® Repair Mix (New England Biolabs) in 20 µL according to the manufacturer's instructions. Repaired product DNA was purified using the QIAEX II system (Qiagen) according to the manufactures instructions and was eluted in 18 µL Milli-Q water. DNA library concentrations were determined using the Quantus™ Fluorometer (Promega). After quality control by electrophoresis with the Bioanalyzer 2100 system (Agilent), DNA libraries were pooled equimolarly and sequencing was performed in paired-end mode on an Illumina MiSeq™ or NextSeq 2000 system with 2 x 75 bp read length. Each prepared NGS library was prepared and sequenced once.