Targeted long-read sequencing facilitates phased diploid assembly and genotyping of the human T cell receptor alpha, delta, and beta loci

Abstract

T cell receptors (TCRs) recognize peptide fragments presented by the major histocompatibility complex (MHC) and are critical to T cell-mediated immunity. Recent data have indicated that genetic diversity within TCR-encoding gene regions is underexplored, limiting understanding of the impact of TCR loci polymorphisms on TCR function in disease, even though TCR repertoire signatures (1) are heritable and (2) associate with disease phenotypes. To address this, we developed a targeted long-read sequencing approach to generate highly accurate haplotype resolved assemblies of the TCR beta (TRB) and alpha/delta (TRA/D) loci, facilitating the genotyping of all variant types, including structural variants. We validate our approach using two mother-father-child trios and 5 unrelated donors representing multiple populations. This resulted in improved genotyping accuracy and the discovery of 84 undocumented V, D, J, and C alleles, demonstrating the utility of this framework for improving our understanding of TCR diversity and function in disease.

Keywords: T cell receptor; TCR; TCR alleles; alpha; beta; delta; immunogenomics; long-read sequencing; long-reads; structural variants.

Graphical abstract

Figure 1

Targeted HiFi sequencing generates highly accurate long reads (A and B) HiFi read (A) lengths and (B) quality. (C ) Coverage of (C) TRA/D and TRB. Each color represents a different sample.

Figure 2

Trio assemblies in TRA/D and TRB are highly accurate (A and B) Examples of high concordance in TRA/D (A) and TRB (B) assemblies are shown between the parents and proband of both trios. The GENCODE (v.24) annotation track is shown.

Figure 3

Genetic variants in TRA/D and TRB (A–C) The per-sample counts of (A) SNPs, (B) indels, and (C) SVs detected from TRA/D and TRB assemblies. (D and E) Comparison of SNPs detected from long-read sequencing (LRS) capture assemblies and 1KGP3 (left) and 1KG-30× (right) variant call sets for (D) TRA/D and (E) TRB; samples with the highest (left) and lowest (right) concordance rates are shown. (F) IGV screenshot showing example region of TRB (chr7:142,438,390–142,547,431), demonstrating the presence of false SNPs identified from the 1KG-30× dataset. The panels shown are (1, top) SNPs present only within the 1KG-30× variant call set, (2) 1KG-30× Illumina 30× 2×151 bp short-read data, (3) PacBio HiFi-reads, and (4, bottom) PacBio HiFi assemblies. The ∼20 kb insertion resolved by the PacBio HiFi reads is highlighted in blue.

Figure 4

Comparison of TRB haplotypes in GRCh38/hg38 and GRGRh37/hg19 (A) Dot plots between TRB loci GRCh38/hg38 chromosome 7, GRCh37/hg19 chromosome 7, and GRCh38/hg38 chr7_KI270803v1_alt. Dot plots were generated using Gepard. Each dot represents sequence homology between the compared sequences. Gaps in the diagonal represent insertions/deletions. Dots perpendicular to the diagonal indicate an inversion. (B) TRB loci from chr7_KI270803v1_alt and GRCh37/hg19 chromosome 7 aligned to the TRB loci from GRCh38/hg38 chromosome 7. Both alignments further demonstrate genetic differences between reference haplotypes. Alignment of chr7_KI270803v1_alt, in particular, identifies three ∼20 kb insertions, indicated by purple marks. The GENCODE (v.24) annotation track is shown. (C) HiFi read coverage in all samples for the chr7_KI270803v1_alt assembly. Shaded regions indicate the three ∼20 kb insertions in chr7_KI270803v1_alt. (D) UCSC genome browser screenshots of chr7_KI270803v1_alt showing genes within the insertions. GENCODE (v.36) annotation track is shown.

Figure 5

TRA/D and TRB allelic diversity Gene alleles resolved from (A) TRA/D and (B) TRB long-read assemblies. Each color represents an allele or a deleted gene allele. Alleles marked by black dots are those not documented in IMGT. Genes with two distinct alleles are indicated by the presence of two filled boxes. Samples are arranged according to the superpopulation labels (EAS, East Asian; AMR, American; AFR, African).