title: 068-EKC-A8C-37c-b-2-p
design: Sequencing libraries were generated using a two-step PCR amplification workflow. First-strand cDNA served as input for PCR1, which was performed using high-fidelity polymerase and locus-specific primers to amplify the target region. PCR1 products were purified with SPRI beads, and a defined amount of purified amplicon was used as input for PCR2. PCR2 incorporated unique dual 8-bp indexes to enable multiplexed sequencing, with indexing primers dispensed into individual wells using an acoustic liquid handler. Following cleanup, individual libraries were quantified by fluorescence-based dsDNA assays and pooled in equimolar ratios. The final pooled library was assessed by Bioanalyzer (High Sensitivity DNA or DNA 1K) prior to sequencing. Libraries were sequenced on an Illumina MiSeq (275 bp) or NextSeq 1000/2000 (2150 bp) platform with a 1015% PhiX spike-in to ensure balanced base composition.