High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias
Abstract
:1. Introduction
2. Results and Discussion
2.1. AlkB and FTO Are Amenable to Phage Display
2.2. High-Throughput Sequencing Reveals Enrichment of Parasitic FTO Genotypes
2.3. Nanopore Sequencing Allows Detection of Genotypes Not Observed by Illumina Sequencing
2.4. Amino Acid Patterns Deduced from Enriched Genotypes Can Be False Positives
2.5. Parasitic Enrichment of FTO Variants Is Enhanced by Amplification Bias
3. Materials and Methods
3.1. AlkB and FTO Phage Production
3.2. Phage Titers
3.3. Anti-C-Myc Phage ELISA
3.4. Library Generation and Biopanning
3.5. Illumina Sequencing
3.6. Oxford Nanopore Sequencing
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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alkB | FTO | ||||||||
---|---|---|---|---|---|---|---|---|---|
Library | Total Paired Reads | CAN (%) | INS9 (%) | Total Paired Reads | CAN (%) | INS25 (%) | INS27 (%) | INS1 (%) | DEL2 (%) |
Naive | 761,541 | 77.09 | 17.22 | 115,191 | 67.69 | 1.06 | 0.14 | 0.01 | 0 |
Round 1 | 151,953 | 82.26 | 14.14 | 106,962 | 28.59 | 19.88 | 0.61 | 0.76 | 0.06 |
Round 2 | 140,577 | 86.26 | 3.71 | 64,156 | 16.7 | 25.24 | 0.71 | 2.6 | 1.67 |
Round 4 | 128,364 | 85.79 | 4.28 | 9453 | 5.74 | 28.49 | 0.51 | 8.05 | 7.39 |
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Plessers, S.; Van Deuren, V.; Lavigne, R.; Robben, J. High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias. Int. J. Mol. Sci. 2021, 22, 5513. https://doi.org/10.3390/ijms22115513
Plessers S, Van Deuren V, Lavigne R, Robben J. High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias. International Journal of Molecular Sciences. 2021; 22(11):5513. https://doi.org/10.3390/ijms22115513
Chicago/Turabian StylePlessers, Sander, Vincent Van Deuren, Rob Lavigne, and Johan Robben. 2021. "High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias" International Journal of Molecular Sciences 22, no. 11: 5513. https://doi.org/10.3390/ijms22115513