We introduce the TRUST4 open-source algorithm for reconstruction of immune receptor repertoires in αβ/γδ T cells and B cells from RNA-sequencing (RNA-seq) data. Compared with competing methods, TRUST4 supports both FASTQ and BAM format and is faster and more sensitive in assembling longereven full-length-receptor repertoires. TRUST4 can also call repertoire sequences from single-cell RNA-seq (scRNA-seq) data without V(D)J enrichment, and is compatible with both SMART-seq and 5′ 10x Genomics platforms.Both T and B cells can generate diverse receptor (TCR and BCR, respectively) repertoires, through somatic V(D)J recombination, to recognize various external antigens or tumor neoantigens. Following antigen recognition, BCRs also undergo somatic hypermutations (SHMs) to further improve antigen-binding affinity. Repertoire sequencing has been increasingly adopted in infectious disease 1 , allergy 2 , autoimmune 3 , tumor immuology 4 and cancer immunotherapy 5 studies, but it is an expensive assay and consumes valuable tissue samples. Alternatively, RNA-seq data contain expressed TCR and BCR sequences in tissues or peripheral blood mononuclear cells (PBMC). However, because repertoire sequences from V(D)J recombination and SHM are different from the germline, they are often eliminated in the read-mapping step.Previously we developed the TRUST algorithm 6-8 , utilized to de novo assemble immune receptor repertories directly from tissue or blood RNA-seq data. When applied to The Cancer Genome Atlas (TCGA) tumor RNA-seq data, TRUST revealed profound biological insights into the repertoires of tumor-infiltrating T cells 6 and B cells 8 , as well as their associated tumor immunity. Although less sensitive than TCR-seq and BCR-seq, TRUST is able to identify the abundantly expressed and potentially more clonally expanded TCRs/BCRs in the RNA-seq data that are more likely to be involved in antigen binding 9 . Recent years have also seen other computational methods introduced for immune repertoire construction from RNA-seq data, including V'DJer 10 , MiXCR 11 , CATT 12 and ImRep 13 . These methods focus on reconstruction of complementary-determining region 3 (CDR3), with limited ability to assemble full-length V(D)J receptor sequences, although CDR1 and CDR2 on the V sequence still contribute considerably to antigen recognition and binding. For example, five out of six mutations predicted in a recent study to influence antibody affinity in the acidic tumor environment are located in CDR1 and CDR2 (ref. 14 ), and four out of nine positions contributing most to 4A8 antibody binding to the SARS-CoV-2 spike protein are in CDR1 and CDR2 (ref. 15 ). Therefore, algorithms that can infer full-length immune receptor repertoires can facilitate better receptor-antigen interaction modeling.

Supplementary data are available at Bioinformatics online.

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BackgroundAcute myeloid leukemia (AML), caused by the abnormal proliferation of immature myeloid cells in the blood or bone marrow, is one of the most common hematologic malignancies. Currently, the interactions between malignant myeloid cells and the immune microenvironment, especially T cells and B cells, remain poorly characterized.MethodsIn this study, we systematically analyzed the T cell receptor and B cell receptor (TCR and BCR) repertoires from the RNA-seq data of 145 pediatric and 151 adult AML samples as well as 73 non-tumor peripheral blood samples.ResultsWe inferred over 225,000 complementarity-determining region 3 (CDR3) sequences in TCR α, β, γ, and δ chains and 1,210,000 CDR3 sequences in B cell immunoglobulin (Ig) heavy and light chains. We found higher clonal expansion of both T cells and B cells in the AML microenvironment and observed many differences between pediatric and adult AML. Most notably, adult AML samples have significantly higher level of B cell activation and more secondary Ig class switch events than pediatric AML or non-tumor samples. Furthermore, adult AML with highly expanded IgA2 B cells, which might represent an immunosuppressive microenvironment, are associated with regulatory T cells and worse overall survival.ConclusionsOur comprehensive characterization of the AML immune receptor repertoires improved our understanding of T cell and B cell immunity in AML, which may provide insights into immunotherapies in hematological malignancies.