TCRpower: quantifying the detection power of T-cell receptor sequencing with a novel computational pipeline calibrated by spike-in sequences

Abstract

T-cell receptor (TCR) sequencing has enabled the development of innovative diagnostic tests for cancers, autoimmune diseases and other applications. However, the rarity of many T-cell clonotypes presents a detection challenge, which may lead to misdiagnosis if diagnostically relevant TCRs remain undetected. To address this issue, we developed TCRpower, a novel computational pipeline for quantifying the statistical detection power of TCR sequencing methods. TCRpower calculates the probability of detecting a TCR sequence as a function of several key parameters: in-vivo TCR frequency, T-cell sample count, read sequencing depth and read cutoff. To calibrate TCRpower, we selected unique TCRs of 45 T-cell clones (TCCs) as spike-in TCRs. We sequenced the spike-in TCRs from TCCs, together with TCRs from peripheral blood, using a 5′ RACE protocol. The 45 spike-in TCRs covered a wide range of sample frequencies, ranging from 5 per 100 to 1 per 1 million. The resulting spike-in TCR read counts and ground truth frequencies allowed us to calibrate TCRpower. In our TCR sequencing data, we observed a consistent linear relationship between sample and sequencing read frequencies. We were also able to reliably detect spike-in TCRs with frequencies as low as one per million. By implementing an optimized read cutoff, we eliminated most of the falsely detected sequences in our data (TCR α-chain 99.0% and TCR β-chain 92.4%), thereby improving diagnostic specificity. TCRpower is publicly available and can be used to optimize future TCR sequencing experiments, and thereby enable reliable detection of disease-relevant TCRs for diagnostic applications.

Reference-based comparison of adaptive immune receptor repertoires

“…Our results reinforce the notion that while some diseases may introduce abnormalities into the immune repertoire, others result in a comparatively normal one ( Bashford-Rogers et al., 2019 ), a result that suggests the absence of a signature unique to health. If this is true, then blood-based immune repertoire diagnostics will require even more advanced methodologies to be developed ( Arnaout et al., 2021 ; Dahal-Koirala et al., 2022 ; Widrich et al., 2020a ). For example, for simulated repertoires, motif implants in ≥10% of sequences were required to affect the amino acid frequency and architecture features, suggesting that even in the case of high clonal expansion, the impact on the repertoire might not be sufficient to significantly change major repertoire features.…”

T-Cell Receptor Repertoire Sequencing and Its Applications: Focus on Infectious Diseases and Cancer

“…Several strategies for experimental validation of TCR recognition are available; we reported as an example the TCR Power [ 2 ], a tool based on spike-in TCRs that estimates the detection probability of a disease-relevant TCR sequence in an experiment through TCR detection power calculated as a function of TCR frequency, TCR sample count, sequencing depth and read cut-off.…”

“…TCR HTS methods can be divided into bulk sequencing, for T-cell populations evaluation or single-cell sequencing for the analysis of individual T-cells [ 2 ]. The choice between these two analyses depends on the goal of the experiment and on other factors, such as sample requirements, hands-on and total workflow time, degree of polymerase chain reaction (PCR) bias, quantifiability, immune repertoire coverage, ease of data analysis and cost.…”

“…Researchers have often performed initial bulk analysis and moved, after selection of features of interest, such as binding affinity, to single-cell [ 45 ], even if recently developed commercial single-cell sequencing solutions start to provide full-length paired αβ-chains sequencing of many T-lymphocytes [ 2 ]. These ultimate technologies are based, for example, on barcoded gel beads mixed with cells, enzymes and partitioning oil, used to generate V(D)J gene expression libraries (e.g., 10X Genomics, Pleasanton, CA, USA).…”

“…Adaptive immunity defense is elicited by large numbers of T-cell receptors (TCRs) and B-cell receptors (BCRs) [ 1 ], and perception and adaptation to external insults enhance pre-existing and generate de novo receptors that the immune system records and retains as immunological memory (e.g., with vaccination) [ 2 ].…”