Deep sequencing analysis of phage libraries using Illumina platform

Abstract

This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of the short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10 7 reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated variable regions from a M13KE phage vector. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2x10 7 reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6 bp), one complimentary region (12 bp) and a variable region (36 bp). We applied this sequencing to a model library of 10 6 unique clones and observed that amplification enriches ~150 clones, which dominate ~20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve diversity of phage clones and identify ligands previously lost in phage display screens.

Phage display revisited: Epitope mapping of a monoclonal antibody directed againstNeisseria meningitidisadhesin A using the PROFILER technology

“…In this respect, our results are in general agreement with those obtained with other methods based on next-generation sequencing of surface-displayed libraries, including phage, 18,19 yeast, 20 or ribosome 21 display. Table S1.…”

“…18 Briefly, gene fragments were expressed in E. coli as either glutathione S-transferase-or Histagged fusions or TRX fusions and purified from the cytoplasmic fraction as soluble forms, as previously described. 18 Recombinant antigens were spotted on nitrocellulose-coated slides (FAST slides, Kerafast, cat. n. BC4182) using the no-contact Marathon Spotter (Arrayjet).…”

“…Signals were considered as positive when their MFI value was higher than 5,000, corresponding to the MFI of protein spots after detection with AlexaFluor Ò 647-conjugated anti-mouse IgG alone, plus 10 standard deviation values. 18 …”

Bacterial community compositions in sediment polluted by perfluoroalkyl acids (PFAAs) using Illumina high-throughput sequencing

“…These conventional molecular biological methods lack sufficient sequences to capture systematic and comprehensive information on various microbial communities (Lu et al 2012). With the development of the secondgeneration sequencing, recently, Illumina high-throughput sequencing technology has confirmed as a highly efficient tool for identifying the entire profile of microbial communities, which can characterize over 10 7 reads in a single running through a paired-end sequencing approach (Matochko et al 2012). This method has been widely used to analyze microbial compositions in various environmental samples, such as wastewater treatment plant (Ma et al 2015), drinking water (Huang et al 2014), and soil (Bartram et al 2011).…”

High throughput functional epitope mapping: Revisiting phage display platform to scan target antigen surface

“…Unbiased mutagenic oligonucleotides having a single triplet coding for each residue would be the solution to achieve optimal sequence space coverage. 60 Beyond the mutagenesis step, library diversity sampling (either before selection or after enrichment on a selector Ab) would greatly benefit from deep sequencing. This procedure is predicted to reveal not only the major sequence features distinguishing groups of Ag variants (recognized or not by the selector mAb), but also minor details contributing to antigenicity that are currently dismissed through sequencing of small sets of clones.…”