Amplicon sequencig o dial-out PCR product usig: Orthogonal PCR  (Set C1)
We rigorously evaluated the efficiency of 81 primers (divided into 48 forward and 33 reverse) by amplifying a high-density pooled library of 150 bp oligos (Twist Bioscience, CA) concurrently using combinations of 12 forward and 11 reverse primers. We indexed all reactions using the NEBNext Multiplex Oligos Primers Set 1 (NEB, catalog no. E7335S), prepared a sequencing library using the NEBNext Ultra II DNA Library Prep Kit (NEB,  catalog no. E7645), and performed paired-end sequencing for 300 cycles using the MiSeq Reagent Kit v2 Micro platform (Illumina, catalog no. MS-102-2002).