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RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Related Experiment Video

Updated: May 20, 2026

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing
12:04

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Published on: October 3, 2018

Deep sequencing analysis of phage libraries using Illumina platform.

Wadim L Matochko1, Kiki Chu, Bingjie Jin

  • 1Department of Chemistry and Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.

Methods (San Diego, Calif.)
|July 24, 2012
PubMed
Summary

Deep sequencing of phage libraries reveals that amplification significantly reduces peptide diversity, potentially causing researchers to miss valuable binding clones. This method enables identification of under-represented clones for improved screening strategies.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Bioinformatics

Background:

  • Phage display is a powerful technique for discovering peptides that bind to specific targets.
  • Traditional Sanger sequencing limits analysis to a few hundred phage clones.
  • Illumina deep sequencing offers high-throughput analysis of millions of reads per run.

Purpose of the Study:

  • To analyze phage-displayed peptide libraries using Illumina deep sequencing.
  • To develop a method for preparing DNA fragments for deep sequencing.
  • To assess the impact of library amplification on peptide diversity.

Main Methods:

  • Preparation of short DNA fragments from M13KE phage vectors using PCR with specialized primers (including barcodes).
  • Illumina HiSeq single-end sequencing to obtain over 2x10^7 reads (57 bp each).
  • Analysis of sequencing data using MatLab software to characterize library diversity and identify enriched clones.

Main Results:

  • Deep sequencing characterized the collapse of diversity in phage libraries due to amplification.
  • Amplification of a model library (10^6 clones) enriched ~150 clones, comprising ~20% of the library.
  • Identified that traditional screening methods may miss thousands of potentially useful clones.

Conclusions:

  • Illumina deep sequencing provides a comprehensive view of phage library diversity, unlike Sanger sequencing.
  • Amplification steps in phage screening can lead to significant loss of valuable peptide diversity.
  • The described deep sequencing approach can identify under-represented clones and inform new screening strategies to preserve diversity.