Abstract
Immune repertoire sequencing of the T-cell receptor can identify clonotypes that have expanded as a result of antigen recognition or hematological malignancies. However, current sequencing protocols display limitations with nonuniform amplification and polymerase-induced errors during sequencing. Here, we developed a sequencing method that overcame these issues and applied it to γδ T cells, a cell type that plays a unique role in immunity, autoimmunity, homeostasis of intestine, skin, adipose tissue, and cancer biology. The ultrasensitive immune repertoire sequencing method used PCR-introduced unique molecular identifiers. We constructed a 32-panel assay that captured the full diversity of the recombined T-cell receptor delta loci in γδ T cells. The protocol was validated on synthetic reference molecules and blood samples of healthy individuals. The 32-panel assay displayed wide dynamic range, high reproducibility, and analytical sensitivity with single-nucleotide resolution. The method corrected for sequencing-depended quantification bias and polymerase-induced errors and could be applied to both enriched and nonenriched cells. Healthy donors displayed oligoclonal expansion of γδ T cells and similar frequencies of clonotypes were detected in both enrichment and nonenriched samples. Ultrasensitive immune repertoire sequencing strategy enables quantification of individual and specific clonotypes in a background that can be applied to clinical as well as basic application areas. Our approach is simple, flexible, and can easily be implemented in any molecular laboratory.
Highlights
The human genome project began on October 1st, 1990, aiming to sequence the human genome
We study patients diagnosed with gastrointestinal stromal tumor (GIST)
The cell-free DNA (cfDNA) is highly fragmented, and thirdly, in early-stage cancer patients, the tumor allele fraction is low. This led to few cellfritt tumör-DNA (ctDNA)-molecules in a sample containing a particular mutation
Summary
The human genome project began on October 1st, 1990, aiming to sequence the human genome. Today the same analysis costs less than $1000 and takes a few days to complete This development has provided us with the ability to detect genomic changes between individuals and helped us to, among other things, understand drug efficiency and the origin of genetic diseases [2]. Further advancements have allowed us to detect genetic changes that occur in individual cells during our lifetime. Somatic mutations have been implicated as having a role in aging and neurodegenerative diseases [3,4]. Another case of somatic variation is our immune system that undergoes genomic scrambling to generate a highly diverse defense system. Studying somatic variation demands an ability to detect extremely low-frequency variations different from the average cell
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