Barcode amplicon preparation for Illumina sequencing

To prepare barcode reads for Illumina sequencing we performed two rounds of PCR. In the first round of PCR we used primers that align to Illumina Truseq Read 1 primer site located directly upstream of the barcode in the lentiviral backbone and a primer annealing downstream of the barcode containing an overhand with Illumina Truseq Read 2 sequence (see ‘Illumina barcode sequencing 1st round PCR primers’ in the Supplemental Table 1). Conditions for the first round PCR were as follows: 1 μl of 10uM forward primer, 1 μl of 10uM reverse primer, 26 μl of KOD, and 24 μl of miniprepped sample DNA. PCR cycling conditions for round 1 PCR were as follows:

PCR reactions were purified with Ampure XP beads using 1:3 sample to beads ratio and eluted in 37 μl of Qiagen elution buffer. Second round of PCR used primers primer annealing to the Illumina Truseq Read 1 primer site with P5 Illumina adapter overhang and reverse primers from the PerkinElmer NextFlex DNA Barcode adaptor set, which anneal to Truseq Read 2 site and contain P7 Illumina adapter and i7 sample index. Conditions for the second round PCR were as follows: 1.5 μl of 10uM universal primer, 1.5 μl of 10uM indexing primer, 25 μl of KOD, and 20 ng of first round PCR product. PCR cycling conditions were the same as the first round PCR for a total of 20 cycles. After the second PCR round all samples were pooled at desired ratios and gel and Ampure XP bead purified. Barcode amplicons were sequenced using NextSeq 2000 with either P2 or P3 reagent kits.