Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
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Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS. / Sloth, Ane Beth; Bakhshinejad, Babak; Stavnsbjerg, Camilla; Rossing, Maria; Kjaer, Andreas.
In: International Journal of Molecular Sciences, Vol. 24, No. 6, 5396, 2023.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
AU - Sloth, Ane Beth
AU - Bakhshinejad, Babak
AU - Stavnsbjerg, Camilla
AU - Rossing, Maria
AU - Kjaer, Andreas
N1 - Publisher Copyright: © 2023 by the authors.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - composition
KW - deep sequencing
KW - distinguishing capacity
KW - diversity
KW - illumina sequencing
KW - next-generation sequencing
KW - Ph.D.-12 peptide library
KW - phage display peptide library
KW - quality
KW - sequencing depth
UR - http://www.scopus.com/inward/record.url?scp=85151111491&partnerID=8YFLogxK
U2 - 10.3390/ijms24065396
DO - 10.3390/ijms24065396
M3 - Journal article
C2 - 36982469
AN - SCOPUS:85151111491
VL - 24
JO - International Journal of Molecular Sciences (Online)
JF - International Journal of Molecular Sciences (Online)
SN - 1661-6596
IS - 6
M1 - 5396
ER -
ID: 343075445