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Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library
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 characterizatio...
Autores principales: | , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742356/ https://www.ncbi.nlm.nih.gov/pubmed/29326697 http://dx.doi.org/10.3389/fimmu.2017.01796 |
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author | Hemadou, Audrey Giudicelli, Véronique Smith, Melissa Laird Lefranc, Marie-Paule Duroux, Patrice Kossida, Sofia Heiner, Cheryl Hepler, N. Lance Kuijpers, John Groppi, Alexis Korlach, Jonas Mondon, Philippe Ottones, Florence Jacobin-Valat, Marie-Josée Laroche-Traineau, Jeanny Clofent-Sanchez, Gisèle |
author_facet | Hemadou, Audrey Giudicelli, Véronique Smith, Melissa Laird Lefranc, Marie-Paule Duroux, Patrice Kossida, Sofia Heiner, Cheryl Hepler, N. Lance Kuijpers, John Groppi, Alexis Korlach, Jonas Mondon, Philippe Ottones, Florence Jacobin-Valat, Marie-Josée Laroche-Traineau, Jeanny Clofent-Sanchez, Gisèle |
author_sort | Hemadou, Audrey |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5742356 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-57423562018-01-11 Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library Hemadou, Audrey Giudicelli, Véronique Smith, Melissa Laird Lefranc, Marie-Paule Duroux, Patrice Kossida, Sofia Heiner, Cheryl Hepler, N. Lance Kuijpers, John Groppi, Alexis Korlach, Jonas Mondon, Philippe Ottones, Florence Jacobin-Valat, Marie-Josée Laroche-Traineau, Jeanny Clofent-Sanchez, Gisèle Front Immunol Immunology 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. Frontiers Media S.A. 2017-12-20 /pmc/articles/PMC5742356/ /pubmed/29326697 http://dx.doi.org/10.3389/fimmu.2017.01796 Text en Copyright © 2017 Hemadou, Giudicelli, Smith, Lefranc, Duroux, Kossida, Heiner, Hepler, Kuijpers, Groppi, Korlach, Mondon, Ottones, Jacobin-Valat, Laroche-Traineau and Clofent-Sanchez. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Immunology Hemadou, Audrey Giudicelli, Véronique Smith, Melissa Laird Lefranc, Marie-Paule Duroux, Patrice Kossida, Sofia Heiner, Cheryl Hepler, N. Lance Kuijpers, John Groppi, Alexis Korlach, Jonas Mondon, Philippe Ottones, Florence Jacobin-Valat, Marie-Josée Laroche-Traineau, Jeanny Clofent-Sanchez, Gisèle Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library |
title | Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library |
title_full | Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library |
title_fullStr | Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library |
title_full_unstemmed | Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library |
title_short | Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library |
title_sort | pacific biosciences sequencing and imgt/highv-quest analysis of full-length single chain fragment variable from an in vivo selected phage-display combinatorial library |
topic | Immunology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742356/ https://www.ncbi.nlm.nih.gov/pubmed/29326697 http://dx.doi.org/10.3389/fimmu.2017.01796 |
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