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

Caitlin A. Kowalsky, Justin R. Klesmith, James A. Stapleton, Vince Kelly, Nolan Reichkitzer, Timothy A. Whitehead

Research output: Contribution to journalArticle

  • 16 Citations

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 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.

LanguageEnglish (US)
Article numbere0118193
JournalPLoS One
Volume10
Issue number3
DOIs
StatePublished - Mar 19 2015

Profile

Practice Guidelines
High-Throughput Nucleotide Sequencing
DNA libraries
Gene Library
Genes
Libraries
Proteins
Gene encoding
genes
amino acid sequences
proteins
experimental design
screening
Phenotype
mutation
phenotype
Design of experiments
Mutation
Screening
DNA

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Kowalsky, C. A., Klesmith, J. R., Stapleton, J. A., Kelly, V., Reichkitzer, N., & Whitehead, T. A. (2015). High-resolution sequence-function mapping of full-length proteins. PLoS One, 10(3), [e0118193]. DOI: 10.1371/journal.pone.0118193

High-resolution sequence-function mapping of full-length proteins. / Kowalsky, Caitlin A.; Klesmith, Justin R.; Stapleton, James A.; Kelly, Vince; Reichkitzer, Nolan; Whitehead, Timothy A.

In: PLoS One, Vol. 10, No. 3, e0118193, 19.03.2015.

Research output: Contribution to journalArticle

Kowalsky, CA, Klesmith, JR, Stapleton, JA, Kelly, V, Reichkitzer, N & Whitehead, TA 2015, 'High-resolution sequence-function mapping of full-length proteins' PLoS One, vol 10, no. 3, e0118193. DOI: 10.1371/journal.pone.0118193
Kowalsky CA, Klesmith JR, Stapleton JA, Kelly V, Reichkitzer N, Whitehead TA. High-resolution sequence-function mapping of full-length proteins. PLoS One. 2015 Mar 19;10(3). e0118193. Available from, DOI: 10.1371/journal.pone.0118193
Kowalsky, Caitlin A. ; Klesmith, Justin R. ; Stapleton, James A. ; Kelly, Vince ; Reichkitzer, Nolan ; Whitehead, Timothy A./ High-resolution sequence-function mapping of full-length proteins. In: PLoS One. 2015 ; Vol. 10, No. 3.
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