title: amplicon sequencing of URA3 and ADE2_biological replicate2
design: genomic DNA of total of 14 populations (Supplemental Dataset. S4) were extracted from cells using the Yeast Genomic DNA Extraction Kit (Solarbio life sciences, Beijing, China) according to the manufacturers instruction. The CRISPR/Cas9 and CRISPR/Cpf1 targeted genomic region of URA3 and ADE2 loci were PCR amplified using 20-bp specific primers locating at 100-bp upstream and downstream of the predicted cleavage site and flanking with 6-bp specific barcode sequence (Supplemental Dataset. S3). And then, the generated 200-bp fragments, regardless of the length of barcode and index sequence, were purified using Gel Extraction Kit (cwbio, Beijing, China) according to the manufacturers instruction. The purified samples were quantified using NanoDrop 2000 (Thermo Scientific, USA), and diluted uniformly to 0.1 g/l. Two l of each dilution was assembled in a library pool and mixed well. The mixed library was sequenced on the Illumina NovaSeq 6000 (Novogene, Tianjin, China) according to the manufacturers protocols. To identify mutations in amplicon sequencing data, MUSCLE (Version 3.8.31, http://www.drive5.com/muscle/) was performed for multiple alignments, and then DNA MAN (version 5.2) software was used to manually proofread.