Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library

Abstract

Phage-display selection of immunoglobulin (IG) or antibody single chain Fragment variable (scFv) from combinatorial libraries is widely used for identifying new antibodies for novel targets. Next-generation sequencing (NGS) has recently emerged as a new method for the high throughput characterization of IG and T cell receptor (TR) immune repertoires both in vivo and in vitro. However, challenges remain for the NGS sequencing of scFv from combinatorial libraries owing to the scFv length (>800 bp) and the presence of two variable domains [variable heavy (VH) and variable light (VL) for IG] associated by a peptide linker in a single chain. Here, we show that single-molecule real-time (SMRT) sequencing with the Pacific Biosciences RS II platform allows for the generation of full-length scFv reads obtained from an in vivo selection of scFv-phages in an animal model of atherosclerosis. We first amplified the DNA of the phagemid inserts from scFv-phages eluted from an aortic section at the third round of the in vivo selection. From this amplified DNA, 450,558 reads were obtained from 15 SMRT cells. Highly accurate circular consensus sequences from these reads were generated, filtered by quality and then analyzed by IMGT/HighV-QUEST with the functionality for scFv. Full-length scFv were identified and characterized in 348,659 reads. Full-length scFv sequencing is an absolute requirement for analyzing the associated VH and VL domains enriched during the in vivo panning rounds. In order to further validate the ability of SMRT sequencing to provide high quality, full-length scFv sequences, we tracked the reads of an scFv-phage clone P3 previously identified by biological assays and Sanger sequencing. Sixty P3 reads showed 100% identity with the full-length scFv of 767 bp, 53 of them covering the whole insert of 977 bp, which encompassed the primer sequences. The remaining seven reads were identical over a shortened length of 939 bp that excludes the vicinity of primers at both ends. Interestingly these reads were obtained from each of the 15 SMRT cells. Thus, the SMRT sequencing method and the IMGT/HighV-QUEST functionality for scFv provides a straightforward protocol for characterization of full-length scFv from combinatorial phage libraries.

Recent Advances in the Molecular Imaging of Atherosclerosis

“…Identification of relevant biomarkers is the stepping stone for an efficient molecular imaging using different modalities. Recent studies highlight the capacity of combining in vivo phage display (using a human combinatorial antibody library) and high-throughput biological screening on atheroma extracts 187 and/or in silico tracking of the most frequently used antibody sequences 188 to select potent and valuable antibody candidates. Further characterization of the targeted molecules by proteomic approaches would give new insights into in vivo targetable, upregulated biomarkers of atherosclerosis.…”

Coupling of Single Molecule, Long Read Sequencing with IMGT/HighV-QUEST Analysis Expedites Identification of SIV gp140-Specific Antibodies from scFv Phage Display Libraries

“…IMGT/HighV-QUEST output files were processed through IMGT statistical and clonotype analysis to determine the number of assigned sequences and clonotypes. The number of sequences assigned to an IMGT clonotype corresponds to in-frame sequences [C104, W118 for VH and C104, F118 for VL (V-KAPPA or V-LAMBDA)] from “1 copy” + “More than 1,” “Single allele” and “Several alleles” (or genes) ( 41 ). Number of in-frame “other categories” sequences were not assigned to a “single gene.” An IMGT clonotype (AA) was defined by a unique V-(D)-J rearrangement (with IMGT gene and allele names determined by IMGT/HighV-QUEST at the nucleotide level) and a unique CDR3-IMGT AA in-frame junction sequence ( 55 , 56 ).…”

“…The screening of this library would be greatly improved with the use of next generation sequencing (NGS) technologies, such as Illumina, however often NGS platforms are limited in the length of the reads (≤400 bp), covering at most one VH or VL domain per read. This limitation has been addressed through the use of alternate high throughput NGS platforms such as the Ion Torrent Personal Genome Machine (PGM) S5 and the Pacific Bioscience (PacBio) RSII and Sequel systems ( 40 , 41 ). Using the PGM-S5 system, He et al generated 900 bp sequencing reads to identify precursors and lineage intermediates of HIV-1 bnAbs from a phage display library.…”

Coupling brain perfusion screens and next generation sequencing to identify blood–brain barrier binding antibodies

“…The use of NGS to follow the progress of combinatorial screening efforts is becoming more widely used. 29,30 Traditionally, it has been very difficult to track round-by-round clonal enrichment during a screen without substantial cost and effort. NGS now enables the monitoring of individual sequences across a screen at a combinatorial level, ultimately aiding in the assessment and success of the screen.…”