PCR Amplicon Purification and Sequencing

We purified the resulting amplicons to remove primer dimers and fragments less than 100bp by binding PCR products to 20uL of Serapure beads [47 (link)] and performing ethanol washes. DNA was subsequently eluted from the beads with 50uL of 10mM Tris pH 8.5. Products were again visualized on a gel to ensure removal of small fragments. Following PCR purification, we prepared libraries for sequencing by attaching unique dual index combinations to DNA from each individual using the Nextera XT indexing kit (Illumina, San Diego, CA), following the manufacturer’s protocol. We then purified the indexed amplicons using Serapure as before. Indexed amplicons were then quantified using a Qubit dsDNA BR Assay (Life Technologies, Carlsbad, CA), visualized using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) and then pooled in equimolar ratios for 2x250 sequencing on an Illumina MiSeq machine (Illumina, San Diego, CA) at the Technology Center for Genomics and Bioinformatics (University of California, Los Angeles).