Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes

Nat Protoc. 2016 Mar;11(3):429-42. doi: 10.1038/nprot.2016.024. Epub 2016 Feb 4.

Abstract

High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typical samples containing >10(4) lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs. Here we present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (>97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Equipment Design
  • High-Throughput Nucleotide Sequencing / economics
  • High-Throughput Nucleotide Sequencing / instrumentation*
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Immunoglobulin Variable Region / genetics*
  • Lab-On-A-Chip Devices / economics
  • Lymphocytes / metabolism*
  • RNA, Messenger / genetics*
  • Receptors, Antigen, T-Cell / genetics*

Substances

  • Immunoglobulin Variable Region
  • RNA, Messenger
  • Receptors, Antigen, T-Cell