Biological controls for standardization and interpretation of adaptive immune receptor repertoire profiling

Abstract

Use of adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread, providing new insights into the immune system with potential broad clinical and diagnostic applications. However, like many high-throughput technologies, it comes with several problems, and the AIRR Community was established to understand and help solve them. We, the AIRR Community’s Biological Resources Working Group, have surveyed scientists about the need for standards and controls in generating and annotating AIRR-seq data. Here, we review the current status of AIRR-seq, provide the results of our survey, and based on them, offer recommendations for developing AIRR-seq standards and controls, including future work.

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

“…Read thresholds are often used in TCR sequencing scenarios to reduce the chance of falsely detected sequences (i.e. false positives), which may be caused by sequencing errors [ 9 ]. Setting the read threshold at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${c}_{\mathrm{thresh}}$\end{document} , the probability of detecting a TCC by receptor sequencing a blood sample is then \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$$ P\left({C}_{\mathrm{read}}>{c}_{\mathrm{thresh}}\right)=1-\sum_{i=0}^{c_{\mathrm{thresh}}}P\left({C}_{\mathrm{read}}=i\right)\qquad\qquad\qquad $$\end{document} \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}\begin{eqnarray*} && =1-\sum_{i=0}^{c_{\mathrm{thresh}}}\sum_{c_{\mathrm{samp}}=1}^{T_{\mathrm{samp}}}{P}_2({C}_{\mathrm{read}} =i|{C}_{\mathrm{samp}}={c}_{\mathrm{samp}},\nonumber\\ && \quad{T}_{\mathrm{read}},{r}_e,\eta, \lambda, {T}_{\mathrm{samp}}){P}_1 ({C}_{\mathrm{samp}}={c}_{\mathrm{samp}}|{f}_{\mathrm{body}},{T}_{\mathrm{samp}})\qquad \end{eqnarray*}\end{document} where the second equation comes from the combined model [ 6 ].…”

“…Several TCR sequencing methods have been developed for the analysis of T-cell populations (bulk sequencing) or of individual T cells (single-cell sequencing) by academics and industrial investigators [ 9 ]. These approaches can be broadly classified into DNA-based or RNA-based approaches, as well as multiplex PCR [using panels of V and J primers (RNA and DNA)] or rapid amplification of 5′ complementary DNA ends (RACE) followed by nested PCR based sequencing (RNA only) [ 9 ]. These different sequencing approaches have their own merits and limitations [ 9 , 10 ] affecting the choice of sequencing approach for different applications.…”

“…These approaches can be broadly classified into DNA-based or RNA-based approaches, as well as multiplex PCR [using panels of V and J primers (RNA and DNA)] or rapid amplification of 5′ complementary DNA ends (RACE) followed by nested PCR based sequencing (RNA only) [ 9 ]. These different sequencing approaches have their own merits and limitations [ 9 , 10 ] affecting the choice of sequencing approach for different applications. Single-cell TCR sequencing provides paired TRA and TRB sequencing, however the number of cells that can be sequenced (10 2 –10 3 ) is much less than bulk TCR sequencing (10 2 –10 6 , 9).…”

“…Recently developed commercial single-cell sequencing solutions (10× genomics) have revolutionized the field by providing full-length paired TRA and TRB sequencing of a large number of T cells. However, bulk TCR sequencing approaches are still typically employed for high-throughput analysis of immune cells in health and disease [ 9 ]. These bulk TCR sequencing approaches have different accuracies and intra- and inter-method reproducibility for detecting TRA and TRB chains [ 11 ].…”

Individualized VDJ recombination predisposes the available Ig sequence space

“…Despite providing a reliable baseline for estimation of the RGMP variation when inferring from samples from the same subject, these types of replicates do not span all steps of AIRR-seq sample generation. For example, our analysis reveals the importance of quantifying the extent to which RGMP differs across different library preparation protocols, for example, RACE, multiplex, and influence of UMI usage (Menzel et al 2014;Khan et al 2016;Vázquez Bernat et al 2019;Barennes et al 2021;Trück et al 2021).…”