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Economical analysis of saturation mutagenesis experiments.

Acevedo-Rocha CG, Reetz MT, Nov Y - Sci Rep (2015)

Bottom Line: In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity.We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters.We show that by carefully considering these parameters, laboratory expenses can be significantly reduced.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Organic Synthesis, Max-Planck-Institut für Kohlenforschung, Mulheim, 45470, Germany [2] Department of Chemistry, Philipps-Universität Marburg, 35032, Germany [3] Prokaryotic Small RNA Biology Group, Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, 35043, Germany [4] Landes-Offensive zur Entwicklung Wissenschafltich-ökonomischer Exzellenz (LOEWE) Centre for Synthetic Microbiology (SYNMIKRO), Philipps-Universität Marburg, 35032, Germany.

ABSTRACT
Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced.

No MeSH data available.


Related in: MedlinePlus

Sequencing results of randomized position S72 (WT codon: AGT) obtained from single colonies formed on agar plates. Vertical axes denote counts (how many codons of each type were found in the sequencing). Black columns denote desalted primers, and grey ones denote HPLC primers. Libraries: (a) 1-2; (b) 3-4; (c) 5-6; (d) 7-8; (e) 9-10; and (f) 11-12.
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f1: Sequencing results of randomized position S72 (WT codon: AGT) obtained from single colonies formed on agar plates. Vertical axes denote counts (how many codons of each type were found in the sequencing). Black columns denote desalted primers, and grey ones denote HPLC primers. Libraries: (a) 1-2; (b) 3-4; (c) 5-6; (d) 7-8; (e) 9-10; and (f) 11-12.

Mentions: The prevalent assumption used in mathematical analyses of SM experiments is that the randomized codon distribution is uniform, i.e., each of the possible codons (32 codons in NNK and in NNS, 22 in 22c-trick, and 20 in Tang) is equally likely to anneal to the template and form a variant42474849. An alternative assumption, which we now examine, is that the distribution is not uniform, and in particular, that there is a bias in favor of the WT codon. Figure 1 shows the distribution of the randomized codons for each of the 12 libraries. Statistical analysis reveals a statistically significant bias in favor of the WT codon, AGT, in five of the libraries: HPLC NNK from supplier 1 (p = 0.004), the two 22c-trick libraries (p = 0.044 for desalted and p < 0.0001 for HPLC), and the two Tang libraries (p = 0.004 for desalted and p < 0.0001 for HPLC). In all of these cases, the randomization resulted in AGT with likelihood significantly higher than expected under a uniform distribution. Since AGT is not among the 32 NNS codons (and indeed, no AGT codon was sequenced in any of the two NNS experiments), the two NNS experiments were excluded from this analysis. In the five remaining libraries, no statistically significant evidence for deviation from uniformity was detected, but the confidence intervals for the probability of WT annealing, PWT, across libraries are too wide and overlapping to rule out the possibility of WT bias also in these cases.


Economical analysis of saturation mutagenesis experiments.

Acevedo-Rocha CG, Reetz MT, Nov Y - Sci Rep (2015)

Sequencing results of randomized position S72 (WT codon: AGT) obtained from single colonies formed on agar plates. Vertical axes denote counts (how many codons of each type were found in the sequencing). Black columns denote desalted primers, and grey ones denote HPLC primers. Libraries: (a) 1-2; (b) 3-4; (c) 5-6; (d) 7-8; (e) 9-10; and (f) 11-12.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507136&req=5

f1: Sequencing results of randomized position S72 (WT codon: AGT) obtained from single colonies formed on agar plates. Vertical axes denote counts (how many codons of each type were found in the sequencing). Black columns denote desalted primers, and grey ones denote HPLC primers. Libraries: (a) 1-2; (b) 3-4; (c) 5-6; (d) 7-8; (e) 9-10; and (f) 11-12.
Mentions: The prevalent assumption used in mathematical analyses of SM experiments is that the randomized codon distribution is uniform, i.e., each of the possible codons (32 codons in NNK and in NNS, 22 in 22c-trick, and 20 in Tang) is equally likely to anneal to the template and form a variant42474849. An alternative assumption, which we now examine, is that the distribution is not uniform, and in particular, that there is a bias in favor of the WT codon. Figure 1 shows the distribution of the randomized codons for each of the 12 libraries. Statistical analysis reveals a statistically significant bias in favor of the WT codon, AGT, in five of the libraries: HPLC NNK from supplier 1 (p = 0.004), the two 22c-trick libraries (p = 0.044 for desalted and p < 0.0001 for HPLC), and the two Tang libraries (p = 0.004 for desalted and p < 0.0001 for HPLC). In all of these cases, the randomization resulted in AGT with likelihood significantly higher than expected under a uniform distribution. Since AGT is not among the 32 NNS codons (and indeed, no AGT codon was sequenced in any of the two NNS experiments), the two NNS experiments were excluded from this analysis. In the five remaining libraries, no statistically significant evidence for deviation from uniformity was detected, but the confidence intervals for the probability of WT annealing, PWT, across libraries are too wide and overlapping to rule out the possibility of WT bias also in these cases.

Bottom Line: In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity.We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters.We show that by carefully considering these parameters, laboratory expenses can be significantly reduced.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Organic Synthesis, Max-Planck-Institut für Kohlenforschung, Mulheim, 45470, Germany [2] Department of Chemistry, Philipps-Universität Marburg, 35032, Germany [3] Prokaryotic Small RNA Biology Group, Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, 35043, Germany [4] Landes-Offensive zur Entwicklung Wissenschafltich-ökonomischer Exzellenz (LOEWE) Centre for Synthetic Microbiology (SYNMIKRO), Philipps-Universität Marburg, 35032, Germany.

ABSTRACT
Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced.

No MeSH data available.


Related in: MedlinePlus