Amplicon sequencing of barcode region of lentiviral genetic barcoding library for viral index 1
To characterize the diversity of viral genetic barcoding libraries, we performed deep sequencing of the barcode region of the lentiviral transfer plasmids. Plasmid was digested using KpnI and MauBI (sites that flank the barcode region) (ThermoFisher) and Ampure XP beads (Beckman Coulter) were used to perform double-sided size selection for the 107 bp product. Sequencing libraries were prepared using the KAPA HyperPrep kit (Roche) following manufacturers instructions using 120 ng of input DNA and an adapter molar ratio of 100:1 (adapter:insert). Product was subjected to double-sided size selection using Ampure XP beads (Beckman Coulter) at bead ratios of 0.5X followed by 2X to purify the target band at ~350 bp and remove residual undigested plasmid. Libraries were sequenced using the Illumina MiSeq platform with >2.7M paired-end 300 bp reads per sample.