title: Highly Accurate Sequencing of Full-Length Immune Repertoire Amplicons Using Tn5-Enabled and Molecular Identifier-Guided Amplicon Assembly.
abstract: Ab repertoire sequencing is a powerful tool to analyze the adaptive immune system. To sequence entire Ab repertoires, amplicons are created from Ab H chain (IgH) transcripts and sequenced on a high-throughput sequencer. The field of immune repertoire sequencing is growing rapidly and the protocols used are steadily improving; however, thus far, immune repertoire sequencing protocols have not been able to sequence full-length immune repertoires including the entire IgH V region and enough of the IgH C region to identify isotype subtypes. In this study, we present a method that combines Tn5 transposase and molecular identifiers for the highly accurate sequencing of amplicons >500 bp using Illumina short read paired-end sequencing. We then apply this method to Ab H chain amplicons to sequence the first, to our knowledge, highly accurate full-length immune repertoire.
authors: Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064.
authors: Charles
authors: C
authors: Cole
authors: Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064.
authors: Roger
authors: http://orcid.org/0000-0002-2898-5150
authors: R
authors: Volden
authors: Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064.
authors: Sumedha
authors: S
authors: Dharmadhikari
authors: Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064.
authors: Camille
authors: C
authors: Scelfo-Dalbey
authors: Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064 vollmers@ucsc.edu.
authors: Christopher
authors: C
authors: Vollmers
mesh_terms: D056704:Adaptive Immunity / Q000235:genetics; D000916:Antibody Diversity; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D056724:Immunity, Humoral / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000502:physiology; D021141:Nucleic Acid Amplification Techniques; D015203:Reproducibility of Results; D019895:Transposases / Q000378:metabolism*
chemical_list: D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D007135:Immunoglobulin Variable Region; C473503:Tn5 transposase; D019895:Transposases