Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
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Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher-throughput (HTP). The capacity of these platforms for characterization of the composition, quality, and diversity of the unselected Ph.D.TM-12 Phage Display Peptide Library was investigated. Our results indicated that HTP sequencing detects a considerably higher number of unique sequences compared to the LTP platform, thus covering a broader diversity of the library. We found a larger percentage of singletons, a smaller percentage of repeated sequences, and a greater percentage of distinct sequences in the LTP datasets. These parameters suggest a higher library quality, resulting in potentially misleading information when using LTP sequencing for such assessment. Our observations showed that HTP reveals a broader distribution of peptide frequencies, thus revealing increased heterogeneity of the library by the HTP approach and offering a comparatively higher capacity for distinguishing peptides from each other. Our analyses suggested that LTP and HTP datasets show discrepancies in their peptide composition and position-specific distribution of amino acids within the library. Taken together, these findings lead us to the conclusion that a higher sequencing depth can yield more in-depth insights into the composition of the library and provide a more complete picture of the quality and diversity of phage display peptide libraries.
Original language | English |
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Article number | 5396 |
Journal | International Journal of Molecular Sciences |
Volume | 24 |
Issue number | 6 |
Number of pages | 13 |
ISSN | 1661-6596 |
DOIs | |
Publication status | Published - 2023 |
Bibliographical note
Publisher Copyright:
© 2023 by the authors.
- composition, deep sequencing, distinguishing capacity, diversity, illumina sequencing, next-generation sequencing, Ph.D.-12 peptide library, phage display peptide library, quality, sequencing depth
Research areas
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