Immune Repertoire Sequencing Using Molecular Identifiers Enables Accurate Clonality Discovery and Clone Size Quantification.

Ben S. Wendel,Ning Jiang,Chenfeng He,Chad M. Williams,Hui Yang,Jun Xiao,Ke-Yue Ma

doi:10.3389/fimmu.2018.00033

Abstract

Unique molecular identifiers (MIDs) have been demonstrated to effectively improve immune repertoire sequencing (IR-seq) accuracy, especially to identify somatic hypermutations in antibody repertoire sequencing. However, evaluating the sensitivity to detect rare T cells and the degree of clonal expansion in IR-seq has been difficult due to the lack of knowledge of T cell receptor (TCR) RNA molecule copy number and a generalized approach to estimate T cell clone size from TCR RNA molecule quantification. This limited the application of TCR repertoire sequencing (TCR-seq) in clinical settings, such as detecting minimal residual disease in lymphoid malignancies after treatment, evaluating effectiveness of vaccination and assessing degree of infection. Here, we describe using an MID Clustering-based IR-Seq (MIDCIRS) method to quantitatively study TCR RNA molecule copy number and clonality in T cells. First, we demonstrated the necessity of performing MID sub-clustering to eliminate erroneous sequences. Further, we showed that MIDCIRS enables a sensitive detection of a single cell in as many as one million naïve T cells and an accurate estimation of the degree of T cell clonal expression. The demonstrated accuracy, sensitivity, and wide dynamic range of MIDCIRS TCR-seq provide foundations for future applications in both basic research and clinical settings.

Highlights

With MID Clustering-based IR-Seq (MIDCIRS) T cell receptor (TCR)-seq, we could achieve about 30% efficiency in recovering the target TCR RNA molecules, which is expected given digital PCR (dPCR) in a nanoliter volume is more efficient than bulk PCR in tubes [36]
The MIDCIRS method offers a flexible strategy for molecular identifiers (MIDs)-barcoded immune repertoire sequencing (IR-seq)
We compared TCR diversity discovered using MIDCIRS with that of MIGEC, using MID with at least two reads as the threshold for both approaches and found that MIGEC led to an underestimated TCR diversity (Figure S8 in Supplementary Material, p < 0.001, effect size r = 0.62)

Summary

Introduction

Immune repertoire sequencing (IR-seq) has become a useful tool to quantify the composition of B or T cell antigen receptor repertoires in basic research, such as vaccination [1,2,3], immune repertoire development [4,5,6,7,8,9], and lymphocyte lineage tracking [2, 9], as well as in various clinical settings, such as minimal residual disease (MRD) monitoring [10], hematopoietic stem cell transplant recovery monitoring [11], and cancer patient prognosis [12, 13]. TCR Clonality Quantification with MIDCIRS diversity and abundance quantification This bottleneck limits the sensitivity of many IR-seq-based assays, such as MRD monitoring. We and others introduced molecular identifiers (MIDs) to IR-seq and DNA/RNA sequencing to reduce errors by tracking each RNA molecule through PCR and sequencing. This approach has significantly improved the accuracy of repertoire profiling [9, 14,15,16,17,18,19], especially to distinguish antibody somatic hypermutations from PCR and sequencing errors. How to use RNA molecular counting to estimate T cell clone size has yet to be established

Methods

Results

Conclusion