1. Marcelo Fernández-Viña
Histocompatibility, Immunogenetics and Disease Profiling Laboratory
Stanford Blood Center

2. Applications of NGS in Immunogenetics
HLA Typing
KIR Typing
Blood Group Typing
Engraftment tests
HLA expression

3. KIR typing
Nelson WC, Pyo CW, Vogan D, Wang R, Pyon YS, Hennessey C, Smith A, Pereira S, Ishitani A, Geraghty DE. An integrated genotyping approach for HLA and other complex genetic systems. Hum Immunol Dec;76(12): doi: /j.humimm PubMed PMID:

4. KIR typing
Selvaraj S, Schmitt AD, Dixon JR, Ren B. Complete haplotype phasing of the MHC and KIR loci with targeted HaploSeq. BMC Genomics Nov 5;16:900. doi: /s PubMed PMID: ; PubMed Central PMCID: PMC

6. KIR
Norman PJ, Hollenbach JA, Nemat-Gorgani N, Marin WM, Norberg SJ, Ashouri E, Jayaraman J, Wroblewski EE, Trowsdale J, Rajalingam R, Oksenberg JR, Chiaroni J, Guethlein LA, Traherne JA, Ronaghi M, Parham P. Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing. Am J Hum Genet Aug 4;99(2): doi: /j.ajhg PubMed PMID: ; PubMed Central PMCID: PMC

7. Figure 1. Pipeline for Analyzing Sequence Data from Highly Polymorphic and Structurally Divergent KIR Haplotypes(A) The PING (Pushing Immunogenetics to the Next Generation) pipeline has two broad arms and two modules. The first module (PING_gc) determines KIR ...
Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham
Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing
null, Volume 99, Issue 2, 2016, 375–391

8. Figure 2. KIR Gene Copy-Number Genotype Determined by Read DepthThe ratio of reads mapping to a specific KIR gene to those mapping to KIR3DL3 can be used for calculating KIR copy number. The results from 97 samples are shown and sorted by ratio. KIR2DL4 (left)...
Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham
Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing
null, Volume 99, Issue 2, 2016, 375–391

9. Figure 3. The KIR Region Is >99
Figure 3. The KIR Region Is >99.99% Covered by Sequence Data(A) Target KIR region on chromosome 19: the gene locations are shown in orange, and pseudogenes are shown in gray. The KIR region varies in gene content, and shown are examples of two frequent A an...
Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham
Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing
null, Volume 99, Issue 2, 2016, 375–391

10. Figure 6. High-Resolution KIR Allele and Copy-Number Genotypes of 97 IHWG CellsFour examples of high-resolution allele and copy-number genotypes of KIR. Individual 1 (SP0010) is homozygous for the KIR A haplotype. Individual 2 (CB6B) has two different B haplot...
Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham
Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing
null, Volume 99, Issue 2, 2016, 375–391

11. Figure 7. Capture of HLA Class I Genes for High-Resolution Allele Genotyping(A) Shown is the read depth across each of the HLA class I genes from a representative sample (chosen by virtue of having a read number closest to the mean number of HLA-specific reads...
Paul J. Norman, Jill A. Hollenbach, Neda Nemat-Gorgani, Wesley M. Marin, Steven J. Norberg, Elham Ashouri, Jyothi Jayaraman, Emily E. Wroblewski, John Trowsdale, Raja Rajalingam, Jorge R. Oksenberg, Jacques Chiaroni, Lisbeth A. Guethlein, James A. Traherne, Mostafa Ronaghi, Peter Parham
Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing
null, Volume 99, Issue 2, 2016, 375–391

12. Blood Groups
Fichou Y, Mariez M, Le Maréchal C, Férec C. The experience of extended blood group genotyping by next-generation sequencing (NGS): investigation of patients with sickle-cell disease. Vox Sang Jul 21. doi: /vox [Epub ahead of print] PubMed PMID:

15. ABO
Lang K, Wagner I, Schöne B, Schöfl G, Birkner K, Hofmann JA, Sauter J, Pingel J, Böhme I, Schmidt AH, Lange V. ABO allele-level frequency estimation based on population-scale genotyping by next generation sequencing. BMC Genomics May 20;17:374. doi: /s PubMed PMID: ; PubMed Central PMCID: PMC

19. ABO Typing
Sample specific barcodes and sequencing adaptors are introduced during PCR, rendering the products suitable for direct sequencing on Illumina MiSeq or HiSeq instruments. Complete sequence coverage of exons 6 and 7 enables molecular discrimination of the ABO subgroups and many alleles. The workflow was applied to ABO genotype more than a million samples. Finally, sequence analysis revealed 287 distinct so far not described alleles of which the most abundant one was identified in 174 samples.

20. Aloisio M, Licastro D, Caenazzo L, Torboli V, D'Eustacchio A, Severini GM, Athanasakis E. A technical application of quantitative next generation sequencingfor chimerism evaluation. Mol Med Rep Oct;14(4): doi: /mmr PubMed PMID: ; PubMed Central PMCID: PMC

21. NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44‑amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (<1%). The protocol followed the standardIon AmpliSeq library preparation and I on Torrent Personal Genome Machineguidelines. Overall, the present study added to the scientific literature, identifying novel technical details for a possible future application of NGS for chimerism quantification.

22. Expression of HLA alleles

23. Nature Genetics 41, (2009)
A genome-wide association study identifies variants in the HLA-DP locus associated with chronic hepatitis B in Asians Yoichiro Kamatani, Sukanya Wattanapokayakit, Hidenori Ochi, Takahisa Kawaguchi, Atsushi Takahashi, Naoya Hosono, Michiaki Kubo, Tatsuhiko Tsunoda, Naoyuki Kamatani, Hiromitsu Kumada, Aekkachai Puseenam, Thanyachai Sura, Yataro Daigo1, Kazuaki Chayama, Wasun Chantratita, Yusuke Nakamura & Koichi Matsuda

24. A Novel Variant Marking HLA-DP Expression Levels Predicts Recovery from Hepatitis B Virus Infection Rasmi Thomasa, Chloe L. Thio, Richard Apps,Ying Qa, Xiaojiang Gao, Darlene Marti, Judy L. Stein, Kelly A. Soderberg, M. Anthony Moody, James J. Goedert, Gregory D. Kirk, W. Keith Hoots, Steven Wolinsky and Mary Carrington
Variation in the 3′ untranslated region of HLA-DPB1 is associated with spontaneous clearance of hepatitis B virus in both Japanese and U.S. populations. The mechanism facilitating viral clearance may be related to the A→G single-nucleotide polymorphism rs , which marks HLA-DP cell-surface expression. The rs G allele is associated with high expression of HLA-DP, and the rs A allele is associated with low expression.
The 496GG genotype, which confers recessive susceptibility to HBV persistence, also associates in a recessive manner with significantly higher levels of HLA-DP surface protein and transcript level expression in healthy donors, suggesting that differences in expression of HLA-DP may increase the risk of persistent HBV infection.

25. HLA-DP surface protein levels correlate with the 496A/G genotype in the 3′UTR region of HLA-DPB1.
HLA-DP surface protein levels correlate with the 496A/G genotype in the 3′UTR region of HLA-DPB1. HLA-DP molecules were detected on peripheral blood CD19+ cells by flow cytometry using the HLA-DP-specific MAb B7/21. Median fluorescence intensity (MFI) of B7/21 staining was compared across 75 normal European-American donors, as grouped by their HLA-DPB1 genotype (A). MFI of staining to the same cells by a pool of Tu39 and SK10 (HLA-II) MAbs that together bind all HLA-DR/DQ/DP allotypes (B) or an isotype-negative control (C) showed no correlation with the 496A/G genotype. P values were generated by unpaired Mann-Whitney t test.
Rasmi Thomas et al. J. Virol. 2012;86:

26. HLA-DPB1 mRNA levels correlate significantly with the 496A/G variant in the 3′UTR region of HLA-DPB1.
HLA-DPB1 mRNA levels correlate significantly with the 496A/G variant in the 3′UTR region of HLA-DPB1. HLA-DPB1 mRNA levels were detected by qPCR amplification of cDNA derived from fresh PBL of 15 healthy European-American donors. HLA-DPB1 mRNA levels were detected using a SYBR green method with primers specific for exon 3 of HLA-DPB1. Levels of HLA-DPB1 mRNA are shown relative to a calibrator using two different reference genes, RPLP0 (A) and GAPDH (B). HLA-DPB1 mRNA levels in the group of individuals with 496AX (n = 10) were significantly lower than that from 496GG (n = 5) donors. These data are representative of three independent experiments. P values were generated by unpaired Mann-Whitney t test.
Rasmi Thomas et al. J. Virol. 2012;86:

27. High HLA-DP Expression and Graft-versus-Host Disease Effie W
High HLA-DP Expression and Graft-versus-Host Disease Effie W. Petersdorf, M.D., Mari Malkki, Ph.D., Colm O’hUigin, Ph.D., Mary Carrington, Ph.D., Ted Gooley, Ph.D., Michael D. Haagenson, M.S., Mary M. Horowitz, M.D., Stephen R. Spellman, M.B.S., Tao Wang, Ph.D., and Philip Stevenson, M.S. N Engl J Med 2015; 373: August 13, 2015DOI: /NEJMoa

28. Fleischhauer K. N Engl J Med 2015;373:669-672.
DPB1 Genomic Structure, Associated DPB1 Single-Nucleotide-Polymorphism Haplotypes, and Resulting HLA-DP Expression.
Figure 1. DPB1 Genomic Structure, Associated DPB1 Single-Nucleotide-Polymorphism Haplotypes, and Resulting HLA-DP Expression. Panel A shows DPB1 genomic structure. Graphic representation of the DPB1 transcript was obtained from the Ensembl Genome Browser ( Dark-blue boxes denote coding exons, light-blue boxes denote untranslated regions (UTRs), and lines connecting the boxes represent introns. The two single-nucleotide polymorphisms (rs , in the 3′ UTR, and rs , in a noncoding region 4989 bp downstream) are indicated in red. The chromosomal location of DPB1 in the cytogenetic ideogram of human chromosome 6 is shown, as is a base-pair scale of the corresponding region. Panel B shows DPB1-rs rs haplotypes as reported by Petersdorf and colleagues.1 DPB1 alleles occurring with a frequency of at least 1% in white persons of European descent are shown in italics. DPB1 alleles in T-cell–epitope group 1 (high immunogenicity) are in red, those in group 2 (intermediate immunogenicity) are in orange, and those in group 3 (low immunogenicity) are in green. HLA-DP expression levels associated with various haplotypes are also depicted.
Fleischhauer K. N Engl J Med 2015;373: 28

29. Petersdorf EW et al. N Engl J Med 2015;373:599-609.
Hazard Ratios for Outcomes of HLA-DPB1 Mismatches in Transplant Recipients, According to the rs Allele Linked to the Mismatch.
Petersdorf EW et al. N Engl J Med 2015;373:

30. Petersdorf EW et al. N Engl J Med 2015;373:599-609.
Probability of Grade II, III, or IV Acute Graft-versus-Host Disease.
Figure 4. Probability of Grade II, III, or IV Acute Graft-versus-Host Disease. The probability of grade II, III, or IV acute graft-versus-host disease (GVHD) is shown for transplant recipients with an rs A-linked HLA-DPB1 mismatch (solid line) or an rs G-linked HLA-DPB1 mismatch (dashed line) when the donor’s HLA-DPB1 was linked to rs A (Panel A) or rs G (Panel B). The effects of the mismatch were deleterious in recipients with rs G-linked HLA-DPB1 (high-expression) only when the donor had rs A-linked (low expression) HLA-DPB1. P values were estimated from multivariable regression models adjusted for age, source of stem cells, disease severity, T-cell depletion (yes or no), transplantation regimen, sex of recipient and donor (male–male, male–female, female–male, or female–female), cytomegalovirus serostatus, and year of transplantation.
Petersdorf EW et al. N Engl J Med 2015;373:

31. Fleischhauer K. N Engl J Med 2015;373:669-672.
DPB1 Genomic Structure, Associated DPB1 Single-Nucleotide-Polymorphism Haplotypes, and Resulting HLA-DP Expression.
Figure 1. DPB1 Genomic Structure, Associated DPB1 Single-Nucleotide-Polymorphism Haplotypes, and Resulting HLA-DP Expression. Panel A shows DPB1 genomic structure. Graphic representation of the DPB1 transcript was obtained from the Ensembl Genome Browser ( Dark-blue boxes denote coding exons, light-blue boxes denote untranslated regions (UTRs), and lines connecting the boxes represent introns. The two single-nucleotide polymorphisms (rs , in the 3′ UTR, and rs , in a noncoding region 4989 bp downstream) are indicated in red. The chromosomal location of DPB1 in the cytogenetic ideogram of human chromosome 6 is shown, as is a base-pair scale of the corresponding region. Panel B shows DPB1-rs rs haplotypes as reported by Petersdorf and colleagues.1 DPB1 alleles occurring with a frequency of at least 1% in white persons of European descent are shown in italics. DPB1 alleles in T-cell–epitope group 1 (high immunogenicity) are in red, those in group 2 (intermediate immunogenicity) are in orange, and those in group 3 (low immunogenicity) are in green. HLA-DP expression levels associated with various haplotypes are also depicted.
Fleischhauer K. N Engl J Med 2015;373: 31

32. Expression of HLA alleles
Define expression variants
Determine variation at the RNA level
NGS may provide additional specificity for further characerization of Genotypes
Possible application for evaluation in tissue expression

33. Summary KIR and Blood Group Typing has been developed
Test Formats and combinations with HLA typing
Quantitation and Expression studies could be applied in the clinical routine setting