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T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing

T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing

Published on: January 12, 2021

Quantitative measurement of pathogen-specific human memory T cell repertoire diversity using a CDR3 beta-specific

Xujing Wang¹, Shuang Jia, Lisa Meyer

¹The Max McGee National Research Center for Juvenile Diabetes, Department of Pediatrics, The Medical College of Wisconsin and The Children's Hospital Research Institute of Children's Hospital of Wisconsin, Milwaukee, WI 53226, USA. xwang@mcw.edu

|September 21, 2007

Summary

This study developed a novel microarray method for precisely quantifying highly similar DNA sequences, crucial for analyzing immune cell repertoires and viral infections with high accuracy.

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Area of Science:

Immunology
Bioinformatics
Molecular Biology

Background:

Quantitative microarray analysis is valuable for complex biological samples with related sequences.
Challenges include accounting for cross-hybridization and varying probe-target hybridization efficiencies.
The T cell repertoire response to influenza peptide was used as a model system.

Purpose of the Study:

To develop a sensitive and accurate microarray method for quantifying complex, highly similar DNA sequences.
To address challenges of cross-hybridization and differential hybridization efficiency in sequence analysis.
To validate the method for analyzing T cell receptor repertoires.

Main Methods:

Designed arrayed probes targeting a 17-nucleotide T cell receptor (TCR)-specific region capable of distinguishing single nucleotide differences.
Normalized hybridization efficiency using a mixture of Cy3-labeled synthetic targets.
Quantified cross-hybridization using the same synthetic targets.
Developed a data handling protocol to correct for hybridization efficiency variations.

Main Results:

The system demonstrated sensitivity to input ratios as low as 0.5% with high accuracy (R2 = 0.81, R = 0.90).
Validation in human subjects showed no significant difference in clonotype abundance ranking compared to traditional cloning and sequencing.
The method successfully analyzed human recall responses to influenza.

Conclusions:

The developed microarray strategy is robust and effective for quantitative resolution of complex, highly similar sequence mixtures.
This approach is adaptable to various applications requiring precise sequence abundance measurement.
The method provides a valuable tool for immune repertoire analysis and other complex sequence studies.