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Nucleic Acids Research 2005 33(11):3667-3677; doi:10.1093/nar/gki669
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Published online 29 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

A computer program for the estimation of protein and nucleic acid sequence diversity in random point mutagenesis libraries

Michael J. Volles* and Peter T. Lansbury, Jr

Center for Neurologic Diseases, Brigham and Women's Hospital and Department of Neurology, Harvard Medical School 65 Landsdowne Street, Cambridge, MA 02139, USA

*To whom correspondence should be addressed. Tel: +1 617 768 8617; Fax: +1 617 768 8606; Email: mvolles{at}rics.bwh.harvard.edu

Received March 31, 2005. Revised May 11, 2005. Accepted June 6, 2005.

A computer program for the generation and analysis of in silico random point mutagenesis libraries is described. The program operates by mutagenizing an input nucleic acid sequence according to mutation parameters specified by the user for each sequence position and type of point mutation. The program can mimic almost any type of random mutagenesis library, including those produced via error-prone PCR (ep-PCR), mutator Escherichia coli strains, chemical mutagenesis, and doped or random oligonucleotide synthesis. The program analyzes the generated nucleic acid sequences and/or the associated protein library to produce several estimates of library diversity (number of unique sequences, point mutations, and single point mutants) and the rate of saturation of these diversities during experimental screening or selection of clones. This information allows one to select the optimal screen size for a given mutagenesis library, necessary to efficiently obtain a certain coverage of the sequence-space. The program also reports the abundance of each specific protein mutation at each sequence position, which is useful as a measure of the level and type of mutation bias in the library. Alternatively, one can use the program to evaluate the relative merits of preexisting libraries, or to examine various hypothetical mutation schemes to determine the optimal method for creating a library that serves the screen/selection of interest. Simulated libraries of at least 109 sequences are accessible by the numerical algorithm with currently available personal computers; an analytical algorithm is also available which can rapidly calculate a subset of the numerical statistics in libraries of arbitrarily large size. A multi-type double-strand stochastic model of ep-PCR is developed in an appendix to demonstrate the applicability of the algorithm to amplifying mutagenesis procedures. Estimators of DNA polymerase mutation-type-specific error rates are derived using the model. Analyses of an alpha-synuclein ep-PCR library and NNS synthetic oligonucleotide libraries are given as examples.


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A. E. Firth and W. M. Patrick
GLUE-IT and PEDEL-AA: new programmes for analyzing protein diversity in randomized libraries
Nucleic Acids Res., July 1, 2008; 36(suppl_2): W281 - W285.
[Abstract] [Full Text] [PDF]


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