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FLAIRR-seq: A novel method for single molecule resolution of near full-length immunoglobulin heavy chain repertoires

Easton E. Ford, David Tieri, Oscar Rodriguez, Nancy Francoeur, Juan Soto, Justin Kos, Ayelet Peres, William Gibson, Catherine A. Silver, Gintaras Deikus, Elizabeth Hudson, Cassandra R. Woolley, Noam Beckmann, Alexander Charney, Thomas C. Mitchell, Gur Yaari, Robert P. Sebra, Corey T. Watson, View ORCID ProfileMelissa L. Smith
doi: https://doi.org/10.1101/2022.09.24.509352
Easton E. Ford
1Department of Microbiology and Immunology University of Louisville School of Medicine, Louisville, Kentucky, USA
2Department of Biochemistry and Molecular Genetics University of Louisville School of Medicine, Louisville, Kentucky, USA
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David Tieri
2Department of Biochemistry and Molecular Genetics University of Louisville School of Medicine, Louisville, Kentucky, USA
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Oscar Rodriguez
2Department of Biochemistry and Molecular Genetics University of Louisville School of Medicine, Louisville, Kentucky, USA
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Nancy Francoeur
3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York City, New York, USA
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Juan Soto
3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York City, New York, USA
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Justin Kos
2Department of Biochemistry and Molecular Genetics University of Louisville School of Medicine, Louisville, Kentucky, USA
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Ayelet Peres
4Faculty of Engineering, Bar Ilan University, 5290002 Ramat Gan, Israel
5Bar Ilan Institute of Nanotechnology and Advanced Materials, Bar Ilan University, 5290002, Ramat Gan, Israel
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William Gibson
2Department of Biochemistry and Molecular Genetics University of Louisville School of Medicine, Louisville, Kentucky, USA
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Catherine A. Silver
2Department of Biochemistry and Molecular Genetics University of Louisville School of Medicine, Louisville, Kentucky, USA
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Gintaras Deikus
3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York City, New York, USA
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Elizabeth Hudson
2Department of Biochemistry and Molecular Genetics University of Louisville School of Medicine, Louisville, Kentucky, USA
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Cassandra R. Woolley
1Department of Microbiology and Immunology University of Louisville School of Medicine, Louisville, Kentucky, USA
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Noam Beckmann
3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York City, New York, USA
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Alexander Charney
3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York City, New York, USA
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Thomas C. Mitchell
1Department of Microbiology and Immunology University of Louisville School of Medicine, Louisville, Kentucky, USA
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Gur Yaari
4Faculty of Engineering, Bar Ilan University, 5290002 Ramat Gan, Israel
5Bar Ilan Institute of Nanotechnology and Advanced Materials, Bar Ilan University, 5290002, Ramat Gan, Israel
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Robert P. Sebra
3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York City, New York, USA
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Corey T. Watson
2Department of Biochemistry and Molecular Genetics University of Louisville School of Medicine, Louisville, Kentucky, USA
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Melissa L. Smith
2Department of Biochemistry and Molecular Genetics University of Louisville School of Medicine, Louisville, Kentucky, USA
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  • ORCID record for Melissa L. Smith
  • For correspondence: ml.smith@louisville.edu
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Abstract

Current Adaptive Immune Receptor Repertoire Sequencing (AIRR-seq) strategies resolve expressed antibody (Ab) transcripts with limited resolution of the constant region. Here we present a novel near full-length AIRR-seq (FLAIRR-Seq) method that utilizes targeted amplification by 5’ rapid amplification of cDNA ends (RACE), combined with single molecule, real-time sequencing to generate highly accurate (>Q40, 99.99%) IG heavy chain transcripts. FLAIRR-seq was benchmarked by comparing IG heavy chain variable (IGHV), diversity (IGHD), and joining (IGHJ) gene usage, complementarity-determining region 3 (CDR3) length, and somatic hypermutation to matched datasets generated with standard 5’ RACE AIRR-seq and full-length isoform sequencing. Together these data demonstrate robust, unbiased FLAIRR-seq performance using RNA samples derived from peripheral blood mononuclear cells, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving novel IG heavy chain constant (IGHC) gene features. FLAIRR-seq data provides, for the first time, simultaneous, single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class-switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk expressed Ab repertoires to date.

Competing Interest Statement

The authors have declared no competing interest.

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FLAIRR-seq: A novel method for single molecule resolution of near full-length immunoglobulin heavy chain repertoires
Easton E. Ford, David Tieri, Oscar Rodriguez, Nancy Francoeur, Juan Soto, Justin Kos, Ayelet Peres, William Gibson, Catherine A. Silver, Gintaras Deikus, Elizabeth Hudson, Cassandra R. Woolley, Noam Beckmann, Alexander Charney, Thomas C. Mitchell, Gur Yaari, Robert P. Sebra, Corey T. Watson, Melissa L. Smith
bioRxiv 2022.09.24.509352; doi: https://doi.org/10.1101/2022.09.24.509352
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FLAIRR-seq: A novel method for single molecule resolution of near full-length immunoglobulin heavy chain repertoires
Easton E. Ford, David Tieri, Oscar Rodriguez, Nancy Francoeur, Juan Soto, Justin Kos, Ayelet Peres, William Gibson, Catherine A. Silver, Gintaras Deikus, Elizabeth Hudson, Cassandra R. Woolley, Noam Beckmann, Alexander Charney, Thomas C. Mitchell, Gur Yaari, Robert P. Sebra, Corey T. Watson, Melissa L. Smith
bioRxiv 2022.09.24.509352; doi: https://doi.org/10.1101/2022.09.24.509352

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