No abstract

The profiling of germline Adaptive Immune Receptor Repertoire (AIRR), including T cell receptors (TR) and immunoglobulin (IG), is medically important but challenging due to high genetic diversity and complex recombination. While databases such as the international ImMunoGeneTics information system (IMGT) provide a valuable collection of AIRR allele sequences, they are far from complete, and many of them lack flanking sequences outside alleles. It will be beneficial to have technologies that can efficiently capture relevant sequences from DNA samples or genome assemblies, perform accurate allele calling, and discover novel alleles. In this study, we developed gAIRR-seq, a probe capture-based targeted sequencing pipeline, to profile genomic sequences of TR and IG from individual DNA samples. We then proposed computational pipelines — gAIRR-call and gAIRR-annotate — to call alleles from gAIRR-seq reads and validate the results with whole-genome assemblies. Genomic DNA samples from Genome in a Bottle (GIAB) reference materials and public whole-genome assemblies were used for benchmarking. We applied gAIRR-call to genotype TRV and TRJ alleles with 100% accuracy and achieved 98% accuracy when considering flanking regions of V alleles. We used gAIRR-annotate to annotate allele positions and collect alleles and flanking regions into databases. gAIRR-annotate validated the alleles using 13 high-quality whole-genome assemblies on 6 samples and discovered 79 novel TRV alleles and 11 novel TRJ alleles. We provided a summary of the number of TR alleles from 13 high-quality draft assemblies with the proposed pipeline. We validated a 65-kbp and a 10-kbp structural variant for HG002 on chromosomes 7 and 14, where TRD and J alleles reside. We also uncovered the disagreement of the human genome GRCh37 and GRCh38 in the TR regions; GRCh37 possesses a 270 kbp inversion and a 10 kbp deletion in chromosome 7 relative to GRCh38.

No abstract

Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution remains poorly defined. We conducted exome sequencing on 89 nonsyndromic BA trios. In 31.5% of the patients, rare and deleterious de novo, homozygous recessive and/or compound heterozygous variants were detected in liver-expressed ciliary genes of diverse ciliary functions. Enrichment of deleterious mutations in liver-expressed ciliary geneset was significant compared to 148 control trios (OR 2.58, 95% CI 1.15-6.07). KIF3B , PCNT and TTC17 are essential for ciliogenesis. Reduced ciliary proteins expression were detected in the BA livers with KIF3B and TTC17 mutations. CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Our findings support a larger genetic contribution to nonsyndromic BA risk than expected. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis.