Next-Generation Sequencing of Phage-Displayed Peptide Libraries

  • Wadim L. Matochko
  • Ratmir DerdaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1248)


Genetically encoded peptide libraries enabled the discovery of ligands for clinically relevant targets and functional materials. Next-generation sequencing (NGS) of these libraries improved the selection of ligands by detecting low abundant clones and quantifying changes in copy numbers of clones without many rounds of selection. Although NGS platforms have been widely used in genome assembly, quantification of gene expression (RNA-seq), and metagenomic analyses, few examples in the literature describe sequencing phage libraries. This chapter aims to provide a detailed method for sequencing a Ph.D.-7 phage display library by Ion Torrent. The main techniques covered in this chapter include (1) preparation of a phage library for sequencing, (2) sequencing, and (3) analysis of the sequencing data by a custom Matlab script.

Key words

Phage display Next-generation sequencing Deep sequencing Ion Torrent Matlab analysis 



The authors thank Sophie Dang and Corey Davis at the Molecular Biology Service Unit for the use of the Ion Torrent Personal Sequencing platform and for helpful advice. This work was supported by funds from the University of Alberta and Alberta Glycomic Centre.


  1. 1.
    Glenn TC (2011) Field guide to next-generation DNA sequencers. Mol Ecol Resour 11:759–769PubMedCrossRefGoogle Scholar
  2. 2.
    Dias-Neto E, Nunes DN, Giordano RJ et al (2009) Next-generation phage display: integrating and comparing available molecular tools to enable cost-effective high-throughput analysis. PLoS One 4:e8338PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Ernst A, Gfeller D, Kan Z et al (2010) Coevolution of PDZ domain-ligand interactions analyzed by high-throughput phage display and deep sequencing. Mol Biosyst 6:1782–1790PubMedCrossRefGoogle Scholar
  4. 4.
    Matochko WL, Chu KK, Jin BJ et al (2012) Deep sequencing analysis of phage libraries using Illumina platform. Methods 58:47–55PubMedCrossRefGoogle Scholar
  5. 5.
    McLaughlin ME, Sidhu SS (2013) Engineering and analysis of peptide-recognition domain specificities by phage display and deep sequencing. In: Keating AE (ed) Methods in protein design, vol 523. Academic, New York, NY, pp 327–349CrossRefGoogle Scholar
  6. 6.
    Ravn U, Gueneau F, Baerlocher L et al (2010) By-passing in vitro screening-next generation sequencing technologies applied to antibody display and in silico candidate selection. Nucleic Acids Res 38:e193PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Ravn U, Didelot G, Venet S et al (2013) Deep sequencing of phage display libraries to support antibody discovery. Methods 60:99–110PubMedCrossRefGoogle Scholar
  8. 8.
    Ryvkin A, Ashkenazy H, Smelyanski L et al (2012) Deep panning: steps towards probing the IgOme. PLoS One 7:e41469PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Staquicini FI, Cardo-Vila M, Kolonin MG et al (2011) Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients. Proc Natl Acad Sci U S A 108:18637–18642PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    ‘t Hoen PAC, Jirka SMG, Ten Broeke BR et al (2012) Phage display screening without repetitious selection rounds. Anal Biochem 421:622–631PubMedCrossRefGoogle Scholar
  11. 11.
    Zhang H, Torkamani A, Jones TM et al (2011) Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems. Proc Natl Acad Sci U S A 108:13456–13461PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Kim T, Tyndel MS, Huang H et al (2012) MUSI: an integrated system for identifying multiple specificity from very large peptide or nucleic acid data sets. Nucleic Acids Res 40:e47PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    D’Angelo S, Mignone F, Deantonio C et al (2013) Profiling celiac disease antibody repertoire. Clin Immunol 148:99–109PubMedCrossRefGoogle Scholar
  14. 14.
    Matochko WL, Cory LS, Tang SKY et al (2013) Prospective identification of parasitic sequences in phage display screens. Nucleic Acids Res 42:1784PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Merriman B, Rothberg JM, Ion Torrent R et al (2012) Progress in Ion Torrent semiconductor chip based sequencing. Electrophoresis 33:3397–3417PubMedCrossRefGoogle Scholar
  16. 16.
    Derda R, Tang SKY, Li SC et al (2011) Diversity of phage-displayed libraries of peptides during panning and amplification. Molecules 16:1776–1803PubMedCrossRefGoogle Scholar
  17. 17.
    Bellot G, McClintock MA, Lin C et al (2011) Recovery of intact DNA nanostructures after agarose gel-based separation. Nat Methods 8:192–194PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Chemistry, Alberta Glycomis CentreUniversity of AlbertaEdmontonCanada

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