<p>Oligonucleotide sequences.</p>

Recent developments in DNA synthesis and sequencing allow the construction of comprehensive gene variant libraries and their functional analysis. Achieving high-replication and thorough mutation characterization remains technically and financially challenging for long genes. Here, we developed an efficient, affordable, and scalable library construction approach that relies on low-cost DNA synthesis and standard cloning technologies, which will increase accessibility to mutational studies and help advance the field of protein science. Each degenerate codon variant is physically associated with multiple DNA barcodes during synthesis, which overcomes the need for long-read sequencing for linking variants to barcodes. We demonstrate the scalability of our approach by constructing a complete library for the multidrug resistance gene PDR1, a 3.2 kb gene encoding a pleiotropic transcription factor in the yeast Saccharomyces cerevisiae. We demonstrate a near-perfect correspondence in the measurement of amino acid variants impact when assessed by barcode sequencing and direct sequencing of the mutated coding sequence.近年来，DNA合成与测序技术的最新进展，使得构建全面的基因变异文库并开展其功能分析成为可能。针对长基因而言，实现高保真扩增与全面的突变特征解析，在技术与成本层面仍颇具挑战。本研究开发了一种高效、经济且可扩展的文库构建方案，该方案依托低成本DNA合成与标准克隆技术，可提升突变研究的可及性，助力蛋白质科学领域的发展。在合成过程中，每个简并密码子变异体（degenerate codon variant）会与多个DNA条形码（DNA barcode）实现物理关联，此举无需借助长读长测序（long-read sequencing）即可完成变异体与条形码的匹配。我们通过为多药耐药基因PDR1构建完整文库，验证了本方案的可扩展性——该基因为酿酒酵母（Saccharomyces cerevisiae）中编码多效性转录因子的3.2 kb基因。研究表明，通过条形码测序与突变编码序列直接测序两种方式评估氨基酸变异体的功能影响时，二者的检测结果近乎完全一致。