Characterize HLA Genes - Pacific Biosciences
Characterize HLA Genes No Imputation Required The PacBio® RS II Sequencing System, with its exceptionally long read lengths, accuracy, and fast turnaround time, is the only system available that can sequence full-length HLA gene in a single read span. Reference Free Consensus generated from contiguous amplicon sequences reliably provides directly phased HLA types without imputation. Sequence through exons 2, 3, 4 and more of the HLA genes for better understanding of disease causality, graft Vs host disease in hematopoietic transplantation research and drug hypersensitivity studies.1,2 • • • •
Phase variations with allele specificity across SNP-poor regions Allele-level segregation without imputation Direct evidence for new alleles through de novo consensus generation Obtain phased information between exons 2-3-4 or sequence full HLA gene for detection of polymorphisms outside of the peptide binding domains • Detect variants in regulatory regions within 5’UTR, intron and 3’UTR for comprehensive biological interpretation of causality association • Sequence 144 full length HLA class I gene amplicons per SMRT® Cell in 2 hours Generate Full-Length Allele Sequence
Validation of Full-Length HLA Class I and Exon 2-4 Long Amplicon HLA Class II Sequencing Across 96 Samples4
PacBio reads Consensus sequence generated by SMRT Analysis
5,432 bp of continuous PacBio consensus read for HLA-A:24:02:01:01 allele3
Locus
No of Expected Alleles
Alleles Discordant to Pre-types
Discordant Alleles Orthogonally Validated
A
175
4
4/4
B
180
3
3/3
4
4/4
Non-validated Discordant
% Concordance
100%
0
100.0
100%
0
100.0
100%
0
100.0
C
175
DRB1
161
9
2
98.8%
DQB1
177
2
-
3
98.3%
Total
868
22
18/18 100%
5
99.4%
7/7
100%
PacBio SMRT Sequencing of 96 samples yielded 863 correctly identified unique allele types (homozygous types were considered single calls). 22 independent calls were discordant to low res UCLA pre-types. 18 of which underwent orthogonal validation using Sanger Sequencing. Orthogonal validation matched 100% of the PacBio discordant calls, demonstrating their accuracy. Four remaining alleles have not been validated yet and one DQB1 allele was missed due to lack of coverage on account of PCR related allele dropout issue.
www.pacb.com/hla
From Targeted Region to Fully Phased, Allele-Specific HLA Typing Assay Design and Library Preparation BC1
Locus specific HLA primer
Universal tag BC2
or
BC3
Customer provided assays
Commercial primers
• Customer-designed or GenDx primers • Multiplex up to 96 (or 384) samples • Library automation solutions available
SMRT® Sequencing on the PacBio RS II • Average read lengths 10-15 kb capable of spanning the majority of HLA Class I and II genes • Sequence both HLA Class I and II genes in 2-3 hours • Serially process up to 16 SMRT Cells in a single run with walkaway automation • Capable of identifying over 16,000 full-length alleles per week, per instrument • Demonstrated consensus accuracies >99.999% (QV 50) by avoiding mapping and systematic errors
Data Analysis with SMRT Analysis and Community Tools
• De novo reference free HLA allele consensus sequence generation in SMRT Analysis • High-quality consensus sequences ready for imputation-free 4-field HLA Typing • Sequence output file formats compatible for commercial HLA typing Software
NGSengine® Software
or
The Assign™ MPS HLA Sequence Analysis Software
Key References 1. Hosomichi K. et al (2013) Phase-defined complete sequencing of the HLA henes by next-generation sequencing. BMC Genomics. 14: 355 2. Marsh, S. GE. (2015) ‘The Challenge of HLA in 2015’. 2015 BMT Tandem meetings, February 11-15, San Diego, Plenary talk in first session, entitled ‘The Rapid Evolution of HLA: Complexities from Primates to Transplantation’ 3. Ranade S, et al. (2013) Allele level sequencing and phasing of full-length HLA class I and II genes using SMRT® sequencing technology. The American Society of Human Genetics Conference (2013 ASHG) Poster Presentation (Abstract 688F). Retrieved April 17, 2015 from https://s3.amazonaws.com/files.pacb.com/pdf/ Allele-Level+Sequencing+and+Phasing+of+Full-length+HLA+Class+I+and+II+Genes+Using+SMRT+Sequencing+Technology++.pdf 4. Ranade S, et al. (2015) Multiplexing Human HLA Class I & II Genotyping with DNA Barcode Adapters for High Throughput Clinical Research. Advances in Genome Biology & Technology Conference (2015 AGBT) Poster Presentation (Abstract 178). Retrieved April 15, 2015 from https://s3.amazonaws.com/files.pacb.com/pdf/ Multiplexing+Human+HLA+Class+I+and+II+Genotyping+with+DNA+Barcode+Adapters+for+High+Throughput+Research%C2%A0.pdf
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2015, 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.pacificbiosciences.com/licenses.html. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell, and Iso-Seq are trademarks of Pacific Biosciences. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx. All other trademarks are the sole property of their respective owners. PN: AP104-050115
www.pacb.com/hla
Phase variations with allele specificity across SNP-poor regions Allele-level segregation without imputation Direct evidence for new alleles through de novo consensus generation Obtain phased information between exons 2-3-4 or sequence full HLA gene for detection of polymorphisms outside of the peptide binding domains • Detect variants in regulatory regions within 5’UTR, intron and 3’UTR for comprehensive biological interpretation of causality association • Sequence 144 full length HLA class I gene amplicons per SMRT® Cell in 2 hours Generate Full-Length Allele Sequence
Validation of Full-Length HLA Class I and Exon 2-4 Long Amplicon HLA Class II Sequencing Across 96 Samples4
PacBio reads Consensus sequence generated by SMRT Analysis
5,432 bp of continuous PacBio consensus read for HLA-A:24:02:01:01 allele3
Locus
No of Expected Alleles
Alleles Discordant to Pre-types
Discordant Alleles Orthogonally Validated
A
175
4
4/4
B
180
3
3/3
4
4/4
Non-validated Discordant
% Concordance
100%
0
100.0
100%
0
100.0
100%
0
100.0
C
175
DRB1
161
9
2
98.8%
DQB1
177
2
-
3
98.3%
Total
868
22
18/18 100%
5
99.4%
7/7
100%
PacBio SMRT Sequencing of 96 samples yielded 863 correctly identified unique allele types (homozygous types were considered single calls). 22 independent calls were discordant to low res UCLA pre-types. 18 of which underwent orthogonal validation using Sanger Sequencing. Orthogonal validation matched 100% of the PacBio discordant calls, demonstrating their accuracy. Four remaining alleles have not been validated yet and one DQB1 allele was missed due to lack of coverage on account of PCR related allele dropout issue.
www.pacb.com/hla
From Targeted Region to Fully Phased, Allele-Specific HLA Typing Assay Design and Library Preparation BC1
Locus specific HLA primer
Universal tag BC2
or
BC3
Customer provided assays
Commercial primers
• Customer-designed or GenDx primers • Multiplex up to 96 (or 384) samples • Library automation solutions available
SMRT® Sequencing on the PacBio RS II • Average read lengths 10-15 kb capable of spanning the majority of HLA Class I and II genes • Sequence both HLA Class I and II genes in 2-3 hours • Serially process up to 16 SMRT Cells in a single run with walkaway automation • Capable of identifying over 16,000 full-length alleles per week, per instrument • Demonstrated consensus accuracies >99.999% (QV 50) by avoiding mapping and systematic errors
Data Analysis with SMRT Analysis and Community Tools
• De novo reference free HLA allele consensus sequence generation in SMRT Analysis • High-quality consensus sequences ready for imputation-free 4-field HLA Typing • Sequence output file formats compatible for commercial HLA typing Software
NGSengine® Software
or
The Assign™ MPS HLA Sequence Analysis Software
Key References 1. Hosomichi K. et al (2013) Phase-defined complete sequencing of the HLA henes by next-generation sequencing. BMC Genomics. 14: 355 2. Marsh, S. GE. (2015) ‘The Challenge of HLA in 2015’. 2015 BMT Tandem meetings, February 11-15, San Diego, Plenary talk in first session, entitled ‘The Rapid Evolution of HLA: Complexities from Primates to Transplantation’ 3. Ranade S, et al. (2013) Allele level sequencing and phasing of full-length HLA class I and II genes using SMRT® sequencing technology. The American Society of Human Genetics Conference (2013 ASHG) Poster Presentation (Abstract 688F). Retrieved April 17, 2015 from https://s3.amazonaws.com/files.pacb.com/pdf/ Allele-Level+Sequencing+and+Phasing+of+Full-length+HLA+Class+I+and+II+Genes+Using+SMRT+Sequencing+Technology++.pdf 4. Ranade S, et al. (2015) Multiplexing Human HLA Class I & II Genotyping with DNA Barcode Adapters for High Throughput Clinical Research. Advances in Genome Biology & Technology Conference (2015 AGBT) Poster Presentation (Abstract 178). Retrieved April 15, 2015 from https://s3.amazonaws.com/files.pacb.com/pdf/ Multiplexing+Human+HLA+Class+I+and+II+Genotyping+with+DNA+Barcode+Adapters+for+High+Throughput+Research%C2%A0.pdf
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2015, 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.pacificbiosciences.com/licenses.html. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell, and Iso-Seq are trademarks of Pacific Biosciences. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx. All other trademarks are the sole property of their respective owners. PN: AP104-050115
www.pacb.com/hla
