TY - JOUR
T1 - High-resolution sequence-function mapping of full-length proteins
AU - Kowalsky, Caitlin A.
AU - Klesmith, Justin R.
AU - Stapleton, James A.
AU - Kelly, Vince
AU - Reichkitzer, Nolan
AU - Whitehead, Timothy A.
N1 - Publisher Copyright:
© 2015 Kowalsky et al.
PY - 2015/3/19
Y1 - 2015/3/19
N2 - 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 fulllength 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 sequencefunction mapping protocols.
AB - 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 fulllength 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 sequencefunction mapping protocols.
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U2 - 10.1371/journal.pone.0118193
DO - 10.1371/journal.pone.0118193
M3 - Article
C2 - 25790064
AN - SCOPUS:84926033357
SN - 1932-6203
VL - 10
JO - PloS one
JF - PloS one
IS - 3
M1 - e0118193
ER -