Amplicon sequencing of human liver genomic DNA using primer 4
PCR was performed with FastStart Taq-DNA-Polymerase (Roche 12032902001) using genomic DNA from human liver cells (Amsbio CD563105). PCR cycle condition: Initial denaturation 95C- 10min, 35 amplification cycles Denaturation 95C- 10sec, Annealing 58C- 15sec and extension 72C- 10sec, and lastly final extension 72C- 5min.  PCR products were analyzed on a 2% agarose gel, yielding bands within the expected size range of approximately 200 bp for each primer pair. Bands were excised from the gel and purified using the QIAquick Gel Extraction Kit (Qiagen, Cat. No./ID: 28704). The gel-extracted PCR products were then subjected to amplicon sequencing. DNA library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit from Illumina (Version 6.0, 3/20), with a starting amount of 1.35 ng of DNA. Libraries were amplified through 20 PCR cycles, profiled using a High Sensitivity DNA assay on an Agilent 2100 Bioanalyzer, and quantified with the Qubit dsDNA HS Assay Kit on a Qubit 2.0 Fluorometer (Life Technologies). All samples were pooled in equimolar ratios, purified using AMPure XP Beads (1.0X ratio), and sequenced on a MiSeq Nano Flowcell using paired-end sequencing (2  159 cycles), with an additional 7 cycles for the index read.