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High-resolution sequence-function mapping of full-length proteins.


ABSTRACT: Comprehensive sequence-function mapping involves detailing the fitness contribution of every possible single mutation to a gene by comparing the abundance of each library variant before and after selection for the phenotype of interest. Deep sequencing of library DNA allows frequency reconstruction for tens of thousands of variants in a single experiment, yet short read lengths of current sequencers makes it challenging to probe genes encoding full-length proteins. Here we extend the scope of sequence-function maps to entire protein sequences with a modular, universal sequence tiling method. We demonstrate the approach with both growth-based selections and FACS screening, offer parameters and best practices that simplify design of experiments, and present analytical solutions to normalize data across independent selections. Using this protocol, sequence-function maps covering full sequences can be obtained in four to six weeks. Best practices introduced in this manuscript are fully compatible with, and complementary to, other recently published sequence-function mapping protocols.

SUBMITTER: Kowalsky CA 

PROVIDER: S-EPMC4366243 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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High-resolution sequence-function mapping of full-length proteins.

Kowalsky Caitlin A CA   Klesmith Justin R JR   Stapleton James A JA   Kelly Vince V   Reichkitzer Nolan N   Whitehead Timothy A TA  

PloS one 20150319 3


Comprehensive sequence-function mapping involves detailing the fitness contribution of every possible single mutation to a gene by comparing the abundance of each library variant before and after selection for the phenotype of interest. Deep sequencing of library DNA allows frequency reconstruction for tens of thousands of variants in a single experiment, yet short read lengths of current sequencers makes it challenging to probe genes encoding full-length proteins. Here we extend the scope of se  ...[more]

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2021-04-22 | GSE172487 | GEO