Journal: The Journal of Molecular Diagnostics : JMD
Article Title: Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies
Figure Lengend Snippet: Evaluation of three different polymerase master mixes in the first and second stages of PCR, for sequencing library preparation. Amplification reaction mixes were assembled with TaqMan genotyping master mix, HotStarTaq Plus master mix, or NEBNext Ultra II Q5 mix during first-stage PCR. All of the first-stage PCR products were assembled in NEBNext Ultra II Q5 master mix ( A ), HotStarTaq Plus master mix ( B ), or TaqMan genotyping master mix ( C ) for second-stage PCR. Libraries were purified with solid-phase reversible immobilization beads and analyzed on an Agilent 2100 DNA bioanalyzer. A 300- to 400-bp target-specific library is indicated by a bracket . Note that the fragments of 100 to 200 bp predominantly contained primer dimers. Green and purple bars indicate lower and upper markers, respectively. All samples were evaluated in triplicate.
Article Snippet: The second stage of PCR was performed in 40 μL volume using 1× NEBNext Ultra II Q5 master mix, HotStarTaq Plus master mix, or 1× TaqMan genotyping master mix; 17 μL of purified product from the first-stage PCR; and 0.5 μmol/L Illumina index primers (San Diego, CA).
Techniques: Polymerase Chain Reaction, Sequencing, Amplification, Purification