Premium
Single‐cell based high‐throughput sequencing of full‐length immunoglobulin heavy and light chain genes
Author(s) -
Busse Christian E.,
Czogiel Irina,
Braun Peter,
Arndt Peter F.,
Wardemann Hedda
Publication year - 2014
Publication title -
european journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.272
H-Index - 201
eISSN - 1521-4141
pISSN - 0014-2980
DOI - 10.1002/eji.201343917
Subject(s) - biology , immunoglobulin light chain , dna sequencing , cloning (programming) , gene , computational biology , single cell sequencing , antibody , genetics , microbiology and biotechnology , phenotype , computer science , programming language , exome sequencing
Single‐cell PCR and sequencing of full‐length I g heavy ( I gh ) and I gk and I gl light chain genes is a powerful tool to measure the diversity of antibody repertoires and allows the functional assessment of B ‐cell responses through direct I g gene cloning and the generation of recombinant m A bs. However, the current methodology is not high‐throughput compatible. Here we developed a two‐dimensional bar‐coded primer matrix to combine I gh and I gk / I gl chain gene single‐cell PCR with next‐generation sequencing for the parallel analysis of the antibody repertoire of over 46 000 individual B cells. Our approach provides full‐length I gh and corresponding I gk / I gl chain gene‐sequence information and permits the accurate correction of sequencing errors by consensus building. The use of indexed cell sorting for the isolation of single B cells enables the integration of flow cytometry and I g gene sequence information. The strategy is fully compatible with established protocols for direct antibody gene cloning and expression and therefore advances over previously described high‐throughput approaches to assess antibody repertoires at the single‐cell level.