High throughput DNA sequencing of genomic DNA samples

Genomic sites of interest were amplified from genomic DNA using the specific primers containing illumina forward and reverse adapters (listed in Supplementary Table). Twenty microliter PCR1 reactions were performed with 0.5 μM of each forward and reverse primer, 1 μl of genomic DNA extract or 300 ng purified genomic DNA, and 10 μl of Phusion Flash PCR Master Mix (Thermo Fisher). PCR reactions were carried out as follows: 98 °C for 10 s, then 20 cycles of [98 °C for 1 s, 55 °C for 5 s, and 72 °C for 10 s], followed by a final 72 °C extension for 3 min. After first round of PCR, unique Illumina barcoding reverse primer were added to each sample in a secondary PCR reaction (PCR 2). Specifically, 20 μl of a PCR reaction contained 0.5 μM of unique reverse Illumina barcoding primer pair and 0.5 μM common forward Illumina barcoding primer, 1 μl of unpurified PCR 1 reaction mixture, and 10 μl of Phusion Flash PCR Master Mix. The barcoding PCR2 reactions were carried out as follows: 98 °C for 10 s, then 20 cycles of [98 °C for 1 s, 60°C for 5 s, and 72 °C for 10 s], followed by a final 72 °C extension for 3 min. PCR 2 products were purified by 1% agarose gel using a QIAquick Gel Extraction Kit (Qiagen), eluting with 15 μl of Elution Buffer. DNA concentration was measured by Bioanalyzer and sequenced on an Illumina MiSeq instrument 150 bp, single-end) according to the manufacturer’s protocols. Alignment of amplicon sequences to a reference sequence and calculation of the A-to-G conversion rate were performed according to reported script¹ (Supplementary Note). To analyze the frequency of bystander and corrected mutation at one CFTR allele, reads were aligned to the reference sequence to the defined editing window, and base calling of two tested bases were carried out simultaneously for each read. To calculate A5G9 or G5G9 frequency, we discarded the reads with low quality (Q < 30) for both of edited bases and used the equation: [frequency of specified point mutation] ÷ [total high-quality reads].