Profiling immunoglobulin repertoires across multiple human tissues by RNA Sequencing

Abstract

Assay-based approaches provide a detailed view of the adaptive immune system by profiling immunoglobulin (Ig) receptor repertoires. However, these methods carry a high cost and lack the scale of standard RNA sequencing (RNA-Seq). Here we report the development of ImReP, a novel computational method for rapid and accurate profiling of the immunoglobulin repertoire from regular RNA-Seq data. ImReP can also accurately assemble the complementary determining regions 3 (CDR3s), the most variable regions of Ig receptors. We applied our novel method to 8,555 samples across 53 tissues from 544 individuals in the Genotype-Tissue Expression (GTEx v6) project. ImReP is able to efficiently extract Ig-derived reads from RNA-Seq data. Using ImReP, we have created a systematic atlas of 3.6 million Ig sequences across a broad range of tissue types, most of which have not been studied for Ig receptor repertoires.We also compared the GTEx tissues to track the flow of Ig clonotypes across immune-related tissues, including secondary lymphoid organs and organs encompassing mucosal, exocrine, and endocrine sites, and we examined the compositional similarities of clonal populations between these tissues. The Atlas of Immunoglobulin Repertoires (The AIR), is freely available at https://github.com/smangul1/TheAIR/wiki , is one of the largest collection of CDR3 sequences and tissue types. We anticipate this recourse will enhance future immunology studies and advance the development of therapies for human diseases. ImReP is freely available at https://github.com/mandricigor/imrep/wiki Results Related workA number of tools have been developed to reconstruct the Ig receptor repertoire.Repertoire analysis from RNA-Seq data typically starts with mapping the reads to the germline V, D, and J genes obtained from the International ImMunoGeneTics (IMGT) database 12 . There are three possible read mapping scenarios: (1) the read is entirely mapped to the V gene; (2) the read is entirely mapped to the J gene; (3) the read is partially mapped to the V and J genes simultaneously. Existing methods consider only reads from category (3). These methods use different underlying algorithms to map reads to germline genes. For example, MiXCR 8 relies on an in-house alignment procedure, IgBlast 13 utilizes BLAST with an optimized set of parameters, and IMSEQ 14 uses in-house pairwise alignment between the read sequence and the germline V and J segment sequences.Following the alignment, MiXCR performs overlapping of previously aligned reads into contigs. The resulting contigs are re-aligned to the V, D, and J genes to verify that the significant portion of non-template N insertions is covered. In contrast to MiXCR, which simultaneously aligns reads to both V, D, and J genes, IgBlast separately aligns the query read to the databases comprised of V, D, and J genes. IgBlast uses a specific sequence to separately align; first, the program finds the best V gene hit. Then, IgBlast masks the aligned read region and performs an alignment to the J gene database. (In the ...

Benchmarking of computational error-correction methods for next-generation sequencing data

“…To generate the BCR-Seq dataset, we have used Immunoglobulin heavy chain (IGH) transcripts. To generate the TCR-Seq dataset, we have used T cell receptor alpha chain (TCRA) 28 . For both BCR-Seq and TCR-Seq we generated samples with read length between 50 and 100bp.…”

DNA Vaccination by Electroporation Amplifies Broadly Cross-Restricted Public TCR Clonotypes Shared with HIV Controllers

“…8A, left). The presence of ADVAX clonotypes was then explored in a second HD dataset reported by S. Mangul and colleagues, and consisting exclusively of public TCR clonotypes (41). The public clonotypes were extracted from RNA-Seq data generated by the Genotype-Tissue Expression Consortium, using samples from 544 individuals, and based on the reconstruction of approximately 200,000 TCR CDR3 sequences.…”

“…More than one third of the Gag293-specific TRAV24 clonotypes from vaccinees were present in the dataset reported by Heather et al (40), and half of those were also present in an independently obtained public clonotype dataset reported by Mangul. et al (41). Multiple Gag293-specific TRAV24 clonotypes could be detected in the healthy repertoire at frequencies above 10 −5 , which is in the high range of T cell precursor frequencies estimated for HIV-derived CD4 epitopes (39, 45, 46).…”

“…Left: % found in a dataset of 541,401 TRA clonotypes and 693,571 TRB clonotypes from healthy donors (HD) (40); Right: % found in a dataset of 4,406 TRA public clonotypes and 3,101 TRB public clonotypes from healthy donors (HD) (41); The percentage of TRAV29 (white bar), TRAV24 (black bar), and TRBV2 (grey bar) Gag293-specific clonotypes found at least once in the HD repertoires is reported.…”

Informatics for Cancer Immunotherapy

“…The original protocols could only sequence unpaired single chains of these receptors; recent protocols make it possible to sequence paired chains [176,177] to give a complete picture of the receptor repertoire. New algorithms allow repertoire sequences to be extracted from DNA-seq or RNA-seq data from bulk tissue [178][179][180] or single cells [181] .…”