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DOI: 10.1101/2021.06.16.448102

Defining protein variant functions using high-complexity mutagenesis libraries and enhanced mutant detection software ASMv1.0

X.Yang A. L. Hong T. Sharpe ...+18 D. E. Root
摘要
Pooled variant expression libraries can test thousands of variants of a gene in a single multiplexed experiment. In a library encoding all single-amino-acid substitutions of a protein, each variant differs from its reference only at a single codon-position located anywhere along the coding sequence, such that identifying these variants by sequencing to read out the outcomes of the screen is a major technical challenge. A popular but expensive brute-force approach is to divide the pool of variants into much smaller sub-libraries in each of which only a small region is mutated to allow direct sequencing readout of variant abundance. Here we present an approach to screen very large variant libraries with mutations spanning a wide region in a single pool, including library design criteria and mutant-detection algorithms that permits reliable calling and counting of variants from large-scale sequencing data.
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