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High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire
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High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire
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Journal Article
High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire
2013
Overview
A new method facilitates high-throughput sequencing of the repertoire of immunoglobulin heavy-light chain pairs in human B cells.
Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V
H
and V
L
) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10
4
capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed
in situ
. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V
H
:V
L
linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V
H
:V
L
pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets—peripheral blood IgG
+
B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.
Publisher
Nature Publishing Group US,Nature Publishing Group
Subject
/ B cells
/ B-Lymphocyte Subsets - cytology
/ B-Lymphocyte Subsets - immunology
/ Biomedical Engineering/Biotechnology
/ Cells
/ High-Throughput Nucleotide Sequencing
/ Humans
/ Immunoglobulin Heavy Chains - blood
/ Immunoglobulin Heavy Chains - genetics
/ Immunoglobulin Light Chains - blood
/ Immunoglobulin Light Chains - genetics
/ Immunoglobulin Variable Region - genetics
/ Influenza Vaccines - administration & dosage
/ Influenza Vaccines - immunology
/ letter
/ Male
/ Neurons
/ Receptors, Antigen, B-Cell - genetics
/ Receptors, Antigen, B-Cell - immunology
/ Vaccines
