Next-Generation Sequencing of Phage-Displayed Peptide Libraries

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.

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