p a c b .c o m / h l a

Locus No of Expected Alleles

Alleles Discordant to Pre-types

Discordant Alleles Orthogonally Validated

Non-validated Discordant % Concordance

A 175 4 4/4 100% 0 100.0

B 180 3 3/3 100% 0 100.0

C 175 4 4/4 100% 0 100.0

DRB1 161 9 7/7 100% 2 98.8%

DQB1 177 2 - 3 98.3%

Total 868 22 18/18 100% 5 99.4%

SMRT Sequencing of 96 samples correctly identified 863 unique allele types and 22 potentially discordant alleles compared to low-res UCLA pre-type data. Orthogonal validation with Sanger Sequencing (of the 18 samples tested) demonstrated 100% of the potential discordant alleles matched the PacBio data. Four remaining alleles have not been validated and one DQB1 allele was miscalled due to PCR-related allele-drop out 3.

5,432 bp continuous PacBio consensus read for HLA-A:24:02:01:01 allele2.

- Phase polymorphisms across SNP-poor regions

- Achieve allele-level segregation

- Detect variants in regulatory regions within 5’ UTRs, introns, and 3’ UTRs

- Characterize full-length transcribed minor variants of HLA alleles

- Obtain direct evidence for new HLA alleles through de novo, reference-free consensus generation

- Flexibly adjust amplicon size (e.g., phase through either exons 2, 3 and/or 4, or the entire HLA gene) or scale project size with cost-effective multiplexing solutions

Single Molecule Real-Time (SMRT®) Sequencing delivers reads that span the lengths of the majority of HLA class I and II genes. Unambiguously phased 4-field HLA types without imputation. With a more accurate and complete picture, gain deeper understanding of immune-related disease causality, graft-versus-host disease in hematopoietic transplantation, and drug hypersensitivity1.

GENERATE FULL-LENGTH ALLELE SEQUENCE VALIDATION OF FULL-LENGTH HLA CLASS I AND EXON 2-4 LONG AMPLICON HLA CLASS II SEQUENCING ACROSS 96 SAMPLESPacBio reads

Consensus sequence generated by SMRT Analysis

FULLY PHASED, ALLELE-SPECIFIC HLA SEQUENCING THE PERFECT PAIR

BC1

BC2

BC3

Universal tag

Locus specific HLA primer

PacBio RS II System

Sequel™ System

KEY REFERENCES1. Hosomichi, K. et al. (2013) Phase-defined complete sequencing of the HLA genes by next-generation sequencing. BMC Genomics. 14, 355 2. Ranade, S. et al. (2013, October) Allele level sequencing and phasing of full-length HLA class I and II genes using SMRT Sequencing technology.

Poster presented at the 63rd Annual Meeting of The American Society of Human Genetics. Boston, MA. 3. Ranade, S. et al. (2015, February) Multiplexing human HLA class I & II genotyping with DNA barcode adapters for high throughput research.

Poster presented at Advances in Genome Biology & Technology Conference. Marco Island, FL. 4. 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. 86(1), 61–63. 5. Hosomichi, K. et al. (2015) The impact of next generation technologies on HLA research. Journal of Human Genetics. Advanced Access. doi:10.1038/

jhg.2015.1026. Mayor, Neema P. et al. (2015) HLA typing for the next generation. PLoS One. 10(5), e0127153. 7. Westbrook, C.C. et al. (2015) No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing.

Human Immunology. doi: 10.1016/j.humimm.2015.03.022

For Research Use Only. Not for use in diagnostic procedures. © Copyright 2016, Pacific Biosciences of California, Inc. All rights reserved. Information in this document is subject to change without notice. Pacific Biosciences assumes no responsibility for any errors or omissions in this document. Certain notices, terms, conditions and/or use restrictions may pertain to your use of Pacific Biosciences products and/or third party products. Please refer to the applicable Pacific Biosciences Terms and Conditions of Sale and to the applicable license terms at http://www.pacb.com/legal-and-trademarks/terms-and-conditions-of-sale/.

Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific Biosciences. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx. Fragment Analyzer is a trademark of Advanced Analytical Technologies. All other trademarks are the sole property of their respective owners. PN: AP104-100616

NGSengine® Software

Commercial primersCustomer provided assays

OR

SMRT Sequencing with PacBio® Systems

- Span the majority of HLA class I and II genes with ~10 kb average read lengths

- Sequence HLA class I and II genes in 2-3 hours

- Identify > 16K full-length alleles per week

- Achieve consensus accuracies > 99.999% by avoiding mapping and systematic errors

Data analysis with SMRT Analysis or SMRT Community tools

- Long Amplicon Analysis (LAA): Generate de novo HLA consensus sequences for 4-field typing

- Analyze data output using commercial HLA typing software from GenDx

Library Preparation

- Utilize off-the-shelf enrichment methods or develop your own assays

- Multiplex amplicons that span partial or full-length HLA genes

- Support available for library automation

FROM TARGETED REGION TO FULLY PHASED, ALLELE-SPECIFIC HLA TYPES