Reveal true HLA allelic diversity
Characterizing polymorphisms in HLA genes outside of the protein binding regions is important for strengthening causality association studies1. High-sequence homology and hyper polymorphisms make it challenging to determine true HLA allelic diversity. Even though sequencing is becoming a more popular tool for HLA typing, diploid ambiguities and a reliance on imputation still persist. This is especially true when the length of homologous sequences between alleles extends beyond the capabilities of most sequencing platforms2.
Access the full spectrum of polymorphism across the entire HLA gene
Gain a deeper understanding of immune-related disease causality, graft-versus-host disease in hematopoietic transplantation, and drug hypersensitivity with Single Molecule, Real-Time (SMRT) Sequencing.
SMRT Sequencing delivers single-molecule observations with 10–15 kb reads capable of spanning the majority of HLA class I and II genes. Generate highly accurate consensus sequences (QV50 demonstrated) from amplicons spanning full-length HLA genes without a reference to obtain directly phased, high-resolution HLA types without imputation2.
Features of this application include the ability to:
- Flexibly scale project size (e.g., phase through either exons 2, 3 and/or 4, or the entire HLA gene) with cost-effective multiplexing solutions
- Phase polymorphisms with allele specificity across SNP-poor regions of HLA genes
- Achieve allele-level segregation without imputation
- Detect variants in regulatory regions within 5’ UTRs, introns, and 3’ UTRs
- Fully characterize minor variants in polyclonal samples, such as cancer or transcripts
- Obtain direct evidence for new HLA alleles through de novo, reference-free consensus generation
Workflow: from targeted region to accurate variant or haplotype
- Library preparation
- SMRT Sequencing on the PacBio Systems
- Data analysis with SMRT Analysis or PacBio DevNet
Featured research: Build complete gene references for HLA class II genes
Immunologists at DKMS generated reference-free consensus sequences of HLA-DPB 1 complete genes. At the time of the study, the IGMT/HLA database contained 12 full-length references out of 550 known HLA-DPB1 alleleles. In a single SMRT Sequencing run, scientists discovered 9 new alleles while generating fully-phased class II HLA genotypes for 48 samples3.
Explore this research further.
To learn more about how SMRT Sequencing powers imputation-free HLA typing, contact us.
- Hosomichi, K., et al., (2015) The impact of next-generation sequencing technologies on HLA research. Journal of Human Genetics. 60 (11), 665–673.
- Nelson, W. C., et al., (2015) An integrated genotyping approach for HLA and other complex genetic systems. Human Immunology. 76 (12), (928-938).
- Lang, K. et al. (September, 2015) Phased full-length SMRT Sequencing of HLA DPB1. Poster presented at 41st Annual Meeting of the American Society for Histocompatibility and Immunogenetics. Savannah, GA..
- Turner, T R et al. (2017) Single molecule real-time (SMRT®) DNA sequencing of HLA genes at ultra-high resolution from 126 International HLA and Immunogenetics Workshop cell lines. HLA
- Chen, Shi-Yi et al. (2017) A transcriptome atlas of rabbit revealed by PacBio single-molecule long-read sequencing. Scientific Reports
- Albrecht, V et al. (2017) Dual redundant sequencing strategy: Full-length gene characterisation of 1056 novel and confirmatory HLA alleles. HLA
- Ashley, Euan A et al. (2016) Towards precision medicine. Nature Reviews. Genetics
- Dahal-Koirala, S et al. (2016) TCR sequencing of single cells reactive to DQ2.5-glia-a2 and DQ2.5-glia-?2 reveals clonal expansion and epitope-specific V-gene usage. Mucosal Immunology
- Gowda, Malali et al. (2016) Comparative analyses of low, medium and high-resolution HLA typing technologies for human populations Journal of Clinical & Cellular Immunology
- Shukla, Sachet A et al. (2015) Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes. Nature Biotechnology
- Hosomichi, Kazuyoshi et al. (2015) The impact of next-generation sequencing technologies on HLA research. Journal of Human Genetics
- Mayor, Neema P et al. (2015) HLA typing for the next generation. PLoS One
- Tilgner, Hagen et al. (2014) Defining a personal, allele-specific, and single-molecule long-read transcriptome. Proceedings of the National Academy of Sciences of the United States of America
- Mostafa, A A et al. (2017) The novel HLA-B*08:183 allele identified by sequence-based typing in a Caucasian leukemia patient. HLA
- Matzaraki, Vasiliki et al. (2017) The MHC locus and genetic susceptibility to autoimmune and infectious diseases. Genome Biology
- Sidahmed, A M E et al. (2017) A novel HLA-B*18 allele, HLA-B*18:124, identified in a German volunteer bone marrow donor. HLA
- Sidahmed, A M Elhassan et al. (2016) HLA-A*23:01:19, a novel variant of HLA-A*23:01, discovered in a West African stem cell transplantation patient. HLA
- Kelleher, J et al. (2016) Identification of the novel B*27:144 allele in an Irish Individual. HLA
- Cereb, Nezih et al. (2015) Advances in DNA sequencing technologies for high resolution HLA typing. Human Immunology
- Hayward, D R et al. (2015) The novel HLA-B*44 allele, HLA-B*44:220, identified by Single Molecule Real-Time DNA sequencing in a British Caucasoid male. Tissue Antigens
- Hayhurst, J D et al. (2015) Two novel HLA alleles, HLA-A*30:02:01:03 and HLA-C*08:113, identified in a South African bone marrow donor. Tissue Antigens
- Poster: McLaughlin, I., et al. (2017) Targeted sequencing using a long-read sequencing technology
- Poster: Pollard, M. O. et al. (2016) The MHC Diversity in Africa Project (MDAP) pilot – 125 African high resolution HLA types from 5 populations
- Poster: Cereb, N et al. (2016) Whole gene sequencing of KIR-3DL1 with SMRT Sequencing and the distribution of allelic variants in different ethnic groups
- Poster: Jenny Gu, Kevin Eng, Anand Sethuraman, Steve Kujawa et al. (2016) Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies
- Poster: Baybayan, Primo et al. (2016) Application specific barcoding strategies for SMRT Sequencing
- Poster: Ranade, Swati et al. (2016) Immune regions are no longer incomprehensible with SMRT Sequencing
- Poster: Schöne, B. et al. (2016) Analysis of 37,000 Caucasian samples reveals tight linkage between SNP RS9277534 and high resolution typing of HLA-DPB1
- Ranade, Swati (2017) Webinar: A Paradigm Shift in HLA Sequencing: From Exons to High-Resolution Allele-Level HLA Typing
- Singh, Siddharth (2017) Webinar: Beginner’s Guide to PacBio SMRT Sequencing Data Analysis
- Cereb, Nezih (2017) AGBT PacBio Workshop: High-throughput HLA class I whole gene and HLA class II long range typing on PacBio RSII and Sequel Platforms
- (2017) Tutorial: Long Amplicon Analysis application
- Pollard, Martin (2016) ASHG Virtual Poster: The MHC Diversity in Africa Project (MDAP) pilot – 125 African high resolution HLA types from 5 populations
- Ashley, Euan (2016) AGBT Roche and PacBio Workshop: Towards precision medicine
- Ranade, Swati (2016) AGBT Virtual Poster: Immune regions are no longer incomprehensible with SMRT Sequencing
- PacBio Certified Service Providers (2017)
- Human Biomedical Research Brochure: The most comprehensive view of the human genome (2017)
- SMRT Sequencing Brochure: Revolutionize genomics with SMRT Sequencing. (2017)
- Application Note: Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies. (2016)