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Next-Generation Sequencing of Phage-Displayed Peptide Libraries

Part of the Methods in Molecular Biology book series (MIMB,volume 1248)

Abstract

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

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  • DOI: 10.1007/978-1-4939-2020-4_17
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Acknowledgments

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.

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Correspondence to Ratmir Derda .

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Appendices

Appendix 1: List of Barcodes Used for Multiplexing

ID Barcode ID Barcode ID Barcode
01 CTAAGGTAAC 18 AGGCAATTGC 35 CTTGAGAATGTC
02 TAAGGAGAAC 19 TTAGTCGGAC 36 TGGAGGACGGAC
03 AAGAGGATTC 20 CAGATCCATC 37 TTGGAGGCCAGC
04 TACCAAGATC 21 TCGCAATTAC 38 TGGAGCTTCCTC
05 CAGAAGGAAC 22 TTCGAGACGC 39 TAAGGCAACCAC
06 CTGCAAGTTC 23 TGCCACGAAC 40 TCCTAACATAAC
07 TTCGTGATTC 24 AACCTCATTC 41 TTGAGCCTATTC
08 TTCCGATAAC 25 CCTGAGATAC 42 CTGGCAATCCTC
09 TGAGCGGAAC 26 TTACAACCTC 43 CCGGAGAATCGC
10 CTGACCGAAC 27 AACCATCCGC 44 CAGCATTAATTC
11 TCCTCGAATC 28 ATCCGGAATC 45 TCTGGCAACGGC
12 TAGGTGGTTC 29 TCGACCACTC 46 TCCTTGATGTTC
13 TCTAACGGAC 30 CGAGGTTATC 47 TTCCTGCTTCAC
14 TTGGAGTGTC 31 TCCAAGCTGC 48 CTGAGTTCCGAC
15 TCTAGAGGTC 32 TCTTACACAC 49 TCCTGGCACATC
16 TCTGGATGAC 33 TTCTCATTGAAC 50 TTCCTACCAGTC
17 TCTATTCGTC 34 TAAGCCATTGTC   

Appendix 2: Optimization of Forward and Reverse Primer Sets

To amplify the phage library region, both forward and reverse strands need to bind at the same conditions. Primer sets of the complementary region were chosen by similarities in their melting temperatures. Table 1 lists the forward and reverse primer sets tested to determine which combination generates the most PCR products with the least impurities.

Table 1 List of primers tested in the amplification of the phage library region

First, all primer combinations (12-bp reverse with 12-bp, 18-bp, and 21-bp forward and 13-bp reverse with 12-bp, 18-bp, and 21-bp forward) were tested at the same annealing temperature (62 °C). The 12-bp reverse primer with 12-bp, 18-bp, and 21-bp forward primers did not result in any PCR product at an annealing temperature of 62 °C. PCR amplification of the forward primers with the 13-bp reverse primer resulted in the PCR product. To determine the optimal annealing temperature, the forward and reverse primer pairs were tested at annealing temperatures from 45 to 65 °C. The primer set containing the 18-bp forward and 13-bp reverse compliment sequences gave the most dsDNA fragment with the least impurities.

Appendix 3: Optimizing Emulsion PCR Conditions

The recommended concentration for emulsion PCR is 26 pM (1.56 × 107 molecules per μL) of dsDNA PCR fragments. 25 μL is added to emulsion PCR to give a final amount of 0.65 pmol. The library region of Ph.D. libraries is small; the total amplicon to be sequenced is 62 bp in a Ph.D.-7 library. Emulsion PCR of this small amplicon can cause polyclonal populations. In order to achieve mostly monoclonal populations, we tested different concentrations of dsDNA in emulsion PCR. It was determined that adding 75 fmol (25 μL of a 3 pM solution) gave sufficient sequencing data (Table 2).

Table 2 Amount of template ISPs after emulsion PCR with different amounts of dsDNA added

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Matochko, W.L., Derda, R. (2015). Next-Generation Sequencing of Phage-Displayed Peptide Libraries. In: Derda, R. (eds) Peptide Libraries. Methods in Molecular Biology, vol 1248. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2020-4_17

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  • DOI: https://doi.org/10.1007/978-1-4939-2020-4_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2019-8

  • Online ISBN: 978-1-4939-2020-4

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