The HLA system. The Application of NGS to HLA Typing. Challenges in Data Interpretation
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- Malcolm Sherman
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1 The Application of NGS to HLA Typing Challenges in Data Interpretation Marcelo A. Fernández Viña, Ph.D. Department of Pathology Medical School Stanford University The HLA system High degree of polymorphism at most of the expressed loci (function) Lack of a single predominant allele, high degree of heterozygosity (function) Strong linkage disequilibrium (unknown, function?) B R D
2 GENOMIC ORGANIZATION OF THE HLA GENES HLA-A HLA-B HLA-C HLA-DQA1 HLA-DQB1 HLA-DRB1 HLA-DPA1 HLA-DPB1 4 DRB1*08 Alleles INTRON 1 INTRON 1 INTRON 1 INTRON 2 2
3 HLA coverage over WGS Coverage A B C DRB1 DQB1 DQA1 DPB1 DPA1 A B C DRB1 DQB1 DQA1 DPB1 DPA1 A B C DRB1 DQB1 DQA1 DPB1 DPA1 A B C Sample 1 Sample 2 Sample 3 DRB1 DQB1 DQA1 DPB1 DPA1 Sample 4 Average Average Average Average Minimum Minimum Minimum Minimum Why not whole-genome sequencing? Inadequate coverage of complex genomic regions, such as HLA. Conventional WGS (30x avg. coverage) provides only sparse coverage of HLA. Complexities due to: Indels GC-rich regions, secondary structure Paralogous genes Repeat regions across HLA loci Cost. Using WGS, to achieve adequate coverage of HLA would require >1,000X avg. coverage J.Immunol Jun 15;148(12): HLA-J, a second inactivated class I HLA gene related to HLA-G and HLA-A. Implications for the evolution of the HLA-A-related genes. Messer G, Zemmour J, Orr HT, Parham P, Weiss EH, Girdlestone J. Ragoussis and co-workers described a class I HLA gene that maps to within 50 kb of HLA-A. Comparison of the nucleotide sequences of HLA-J alleles shows this gene is more related to HLA-G, A, and H. All alleles of HLA-J are pseudogenes because of deleterious mutations that produce translation termination either in exon 2 orexon 4. HLA-J appears, like HLA-H, to be an inactivated gene that result from duplication of an Ag-presenting locus related to HLA-A. Evolutionary relationships as assessed by construction of trees suggest the four modern loci: HLA-A, G, H, and J were formed by successive duplications from a common ancestral gene. In this scheme one intermediate locus gave rise to HLA-A and H, the other to HLA-G and J. 3
4 Alleles at different HLA loci (genes and pseudogenes) share nucleotide sequences HLA_A and HLA-H (pseudogene) AA Codon A*24:02:01:01 GAC CGG GCC ATG CCG GGC TAC GTG GAC ACG CAG TTC GTG TTC GAC AGC GAC GCG AGC CAG AGG GAG CCG CGG GCG A A*01:01:01:01 A*02:01:01: A*25:01: A*32:01: T H*01:01:01:01 GGC TAC GTG GAC GAT ACG CAG TTC GTG CGG TTC GAC AGC GAC GCC GCG AGC CAG AGG ATG GAG CCG CGG GCG CCG HLA-A, B and HLA-H (pseudogene) AA Codon B*57:01:01 ATC TAC GCC GAG AAC CTG CGG GCG CTC CGC TAC AAC CAG AGC GAG G --- -G A- CT- -G- G B*07:02:01 B*08:01: G A- CT- -G- G B*15:17:01: B*35:01:01: G A- CT- -G- G B*44:02:01: C -C B*51:01:01: AA Codon H*01:01:01:01 ATC TAC GGC GAG AAC CTG CGG GCG CTC CGC TAC AAC CAG AGC GAG G AA Codon A*24:02:01:01 ATC TAC GCC GAG AAC CTG CGG GCG CTC CGC TAC AAC CAG AGC GAG G -C G-- -C- CT- -G- G A- - A*01:01:01:01 A*02:01:01:01 -T- G G-- -C- CT- -G- G A*25:01: G A- - A*32:01: G DRB Gene Content varies in Haplotypes Bearing Different DRB1 Allele-Sero-Groups (Copy Number Variation has been known in HLA for more than 3 decades) Haplo-groups DR1 DQB1 DQA1 DRB1 DRB6 DRB9 DRA HLA-B DR51 DQB1 DQA1 DRB1 DRB6 DRB5 DRB9 DRA HLA-B DR52 DQB1 DQA1 DRB1 DRB2 DRB3 DRA HLA-B DR8 DQB1 DQA1 DRB1 DRB9 DRA HLA-B DR53 DQB1 DQA1 DRB1 DRB7 DRB8 DRB4 DRB9 DRA HLA-B Nature Jul 3-9;322(6074): Polymorphism of human Ia antigens: gene conversion between two DR beta lociresults in a new HLA-D/DR specificity. Gorski J, Mach B. Molecular mapping of the DR beta-chain region allows true allelic comparisons of the two expressed DR beta-chain loci, DR beta I and DR beta III. At the more polymorphic locus, DR beta I, the allelic differences are clustered and may result from gene conversion events over very short distances. The gene encoding the HLA-DR3/Dw3 specificity has been generated by a gene conversion involving the DR beta I and the DR beta III loci of the HLA-DRw6/Dw18 haplotype, as recipient and donor gene, respectively. The generation of HLA-DR polymorphism within the DRw52 supertypic group can thus be accounted for by a succession of gene duplication, divergence and gene conversion. 4
5 Alleles at different HLA-DRB loci share nucleotide sequences AA Codon DRB1*01:01:01 CAG TGT GGG CGG CA CGT TTC TTG TGG CTT AAG TTT GAA CAT TTC TTC AAT ACG GAG CGG GTG TTG CTG GAA AGA DRB1*01:03 DRB1*03:01:01: GA- T-C TC- -C- -C- --G AC C --- DRB1*04:02: GA- --- G-- --A CA- --G C C C --- DRB1*07:01:01: C GG A- A-G C A- --C DRB1*11:01: GA- T-C TC- -C- -C- --G C C --- DRB1*11: GA- -T C- --G C C --- DRB1*13:01: GA- T-C TC- -C- -C- --G C C --- DRB3*01:01:02: GA- -T- -G C- --G AC C --- DRB3*02:02:01: GA- -T C- --G C G --- Alleles at different HLA-DRB loci share nucleotide sequences Importance of determining Phase AA Codon DRB1*01:01:01 CA CGT TTC TTG TGG CAG CTT AAG TTT GAA TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGG TTG CTG GAA AGA DRB1*03:01:01: GA- T-C TC- -C- -C- --G AC C --- DRB1*13:01:01: GA- T-C TC- -C- -C- --G C C --- DRB1*13: GA- -T C- --G C C --- DRB3*01:01:02: GA- -T- -G C- --G AC C --- DRB3*02:02:01: GA- -T C- --G C G --- AA Codon DRB1*01:01:01 TGC ATC TAT AAC CAA GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TAC CGG GCG GTG ACG GAG CTG GGG DRB1*03:01:01:02 -A- T-- C G AA T DRB1*13:01:01:01 -A- T-- C G AA T DRB1*13:67 -A- T-- C G AA T DRB3*01:01:02:01 -A- T-- C G T- C DRB3*02:02:01:02 CA- T-- C G A- -C G AA Codon DRB1*01:01:01 CGG CCT GAT GCC GAG TAC TGG AAC AGC CAG AAG GAC CTC CTG GAG CAG AGG CGG GCC GCG GTG GAC ACC TAC TGC DRB1*03:01:01: A G- CG AT DRB1*13:01:01: A A G-C GA DRB1*13: A A G-C GA DRB3*01:01:02: TC C A G- CG AT DRB3*02:02:01: A G- CA AT AA Codon DRB1*01:01:01 AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA GTG CAG CGG CGA G DRB1*03:01:01: TG DRB1*13:01:01: TG DRB1*13: DRB3*01:01:02: DRB3*02:02:01: HLA typing using high throughput sequencing technologies. Exon-wise amplification of few exons. Whole-gene amplification. 15 5
6 16 Sequencing workflow Ex 1 Ex 2 Ex 3 Ex 4 Fragmentation Ligate barcoded adaptors Size select and purify bp fragments Sequencing library Q/C Ex 1 Ex 2 Ex 3 Ex 4 6
7 What would be the ideal sequencing machine? High-throughput Accurate Long read length Simple to use Able to detect all types of genomic changes (SNP s, insertion or deletionss, large scale rearrangements, methylation) 19 High-throughput sequencing technologies: an overview All platforms share core similarities: DNA templates are spatially segregated, no physical separation step DNA is sequenced through synthesis, rather than termination DNA sequence is decoded by the emission of light or ph change Platforms differ by: Specific method used to generate template libraries Chemistries/approaches used to generate the sequence signal (light) signal Throughput (amount of bases sequenced per run) Length of sequence read Error modalities and error rates (e.g. homopolymer regions) 20 The Platforms that we Tested 454-Roche: exon coverage only Multiplexing Work flow was demanding ( ) PGM-Ion Torrent: Instrument problems reads too short - homopolymer problems ( ) Pacific Biosciences: Extremely log reads!-throughput and Workflow still in development; appears to be simple Base calling (15 percent error) by consensus -homopolymer problems (2014) Illumina: Less error rate robust instruments ( ). Various systems 7
8 Examples of ambiguities: exon shuffling, segmental exchange, substitutions in untested segments Potential benefits of next-generation sequencing for HLA typing Clonal template amplification in vitroto eliminate problem of sequencing heterozygous DNA Sufficiently long read length (300+ bp) to cover entire exon (or more) in phase Increased sequence coverage of HLA genes Capability to multiplex patient specimens Potential to complete run and data analysis within one week 23 Practical Advantages or of Extending Sequence Coverage Test complete gene No Assumptions made Transplantation: Detect mismatches thought to be absent Mapping of Disease Susceptibility Factors 8
9 Many allele groups in HLA-A show one allele with an insertion of an extra C after seven C A* AC CCC CCC.AAG ACA CAT ATG ACC CAC CAC A*0104N C A* G A --G T A* A*0321N C A* G T A*3114N C--- --G T C A* AAAACGCATATGACTCACCAC A*0321N CAAGACACATATGACCCACCA MAARMSMMWWWK A* AAGACACATATGACCCACCAC A*02null CAAAACGCATATGACTCACCA MARAMMSMWWWK Resolution of common and well documented null- alleles ( clinically relevant) Locus Allele related allele Difference Change Resolution Alternative A 0104N EXON 4 ins 1 routine SBT A 0253N EXON 2 PTC routine SBT A 2409N EXON 4 PTC routine SBT A 2411N EXON 4 ins 1 routine SBT A 6811N EXON 1 del 1 ad hoc SSP B N INTRON 1 del 10 ad hoc SSP extend reading by SBT B 4022N EXON 3 PTC routine SBT B 4423N EXON 3 PTC routine SBT B 5111N EXON 4 ins 1 routine SBT Cw 0409N EXON 7 del 1 ad hoc SSP Cw 0507N EXON 3 del 2 routine SBT DRB N INTRON 1 splicing site ad hoc SSP extend reading by SBT DRB5 0108N 0102 EXON 3 del 19 ad hoc SSP DRB5 0110N 0102 EXON 2 del 2 routine SBT del = nuc. deletion ins = nuc. insertion PTC = premature termination codon Cw*0401/Cw*0409N if B*4403 is present DRB5*0102/0108N if possible haplotype is DRB1*1502-DQB1* Detection of C*04:09N (common) and A*31:14N(rare) allele in single pass A*31:01:02 (red line) shows interrupted coverage at the beginning of Exon 4, while A*31:14N (blue line), which differs from A*31:01:02 with one base insertion, show continuous coverage. C*04:01:01:01 (red line) shows interrupted coverage at the end of Exon 7, while C*04:09N(blue line), which differs from C*04:01:01:01 with one base deletion, show continuous coverage. 9
10 HLA Typing by NGS Wang C, Krishnakumar S, Wilhelmy J, Babrzadeh F, Stepanyan L, Su LF, Levinson D, Fernandez-Viña MA, Davis RW, Davis MM, Mindrinos M High-throughput, high-fidelity HLA genotyping with deep sequencing. Proc Natl Acad Sci U S A. 2012May 29;109(22): doi: /pnas Epub 2012 May 15. PubMed PMID: ; PubMed Central PMCID: PMC New methodology that leverages the power of Next Generation Sequencing (NGS) and long range PCR Interrogated the entire sequences of the class I genes and most of the extent Class II genes in more than 9,000 subjects NGS HLA TYPING SYSTEMS 7. Data analysis 1. Sample Collection 5 UTR UTR HLA-A 5 UTR UTR HLA-B 6. Sequencing 5 UTR UTR 5 UTR UTR 5 UTR UTR HLA-C HLA-DQA1 HLA-DQB1 2. Long-Range PCR 5 UTR UTR HLA-DPB1 5 UTR UTR HLA-DPA1 5 UTR UTR HLA-DRB1, 3, 4, 5 5. Library preparation & Pooling 4. Fragmentation 3. Quantification & Pooling Data Analysis Shotgun sequencing 30 10
11 Genotype calling Coverage Genomic mapping A*02:01:01: A*02: Position Coverage cdna mapping A*02:01:01: A*02: Position One nucleotide difference at exon 3 distinguishes A*02:01:01:01(A) from A*02:07(G). The cell line BM9 HLA-A is A*02:01:01:01. Top left pane shows the coverage plot when sequencing reads are mapped to A*02:01:01:01 and A*02:07 genomicsequence. Top right panel shows the coverage plot when sequencing reads are mapped to A*02:01:01:01 and A*02:07 cdna sequence. High-throughput, High resolution HLA genotyping 32 Data Analysis Steps De-multiplexing Identical barcodes at both ends of pair-end reads Lowering the chance of cross-contamination Mapping Competitive mapping All available reference sequence, including those form pseudo-genes are mapped, best alignments are passed. Filtering Best alignments identical alignments (for cdna only) Pair-end alignment Genotype calling Limited number of candidates (top 10 of each category: number of reads mapped, minimal coverage, minimal central coverage) Enumerate possible combination of homozygous and heterozygous set Rank those combination on aggregated number of reads mapped, minimal coverage, minimal central coverage. De novo Assembly Local de novo assembly can be performed to capture SNP for novel allele 33 11
12 Paired-end Sequencing Pair-end reads Reference Sequence 1 Reference Sequence 2 ~500bp 34 Central Read Definition 35 Using Central Reads Coverage On regular coverage plot, the two candidates looks similar. On central read coverage plot, the wrong candidate have much lower coverage in comparison with the authentic candidate
13 Complement Logics Resolved Difficult Alleles C*03:03:01 and C*03:04:01:01 differ in a single base at the end of exon 2. Due to similarity between some B alleles and C alleles at this region, with cdna alignment, there is no much difference between those two candidates. With genomic alignment and paired-end filter, the difference between those two candidates is greatly amplified to provide definite evidences to call one versus the other. 37 Using Complement Logics cdna alignment genomic alignment Some short exons such as exon 6 of some C alleles are identical to that of B alleles. With cdna alignment, it is hard to predict whether the alignment is authentic. With genomic alignment and pairend reads, the neighboring polymorphic site provides sufficient information for this. 38 Level of parallelism Maximize Usage of Computing Power for Speed Raw reads One process * B1 B2 B3 B4 B5 De-multiplexing M-processes per barcode ***** B1.1 B1.2 B1.3 B1.4 B1.5 Mapping One processes per barcode *** B1 Merging, Filtering, De-multiplexing B1.D B1.D Several processes per barcode ***** B1.A B1.B B1.C PA PB Genotype calling Streaming SIMD Extensions-vectorized implementation of Smith-Waterman algorithm 39 13
14 User Friendly Interface Data analysis pipeline runs with one single command: hla_pipeline.py c config.file Result reviewing is through web page graphically. The two components will be merged together in a single standalone program in next about 6 months. 40 Interface Counting Logics Sample Info Genotypes Candidate Commenting 41 Interface Coverage plot Central Read Coverage plot Reference alignment Read tiling pattern 42 14
15 Phasing Strategy de novo Assembly Multiple fragments of similar sequences generated by NGS GCCAATGATGCACTGACTAGCCTAGCCACCC GCCAATGATGCACTGACTAGCCTAGCCACCC GCCAATGATGCACTGACTAGCCTAGCCACCC TGCACTGACTAGCCTAGCCACCCGATCAGCTCC TGCACTGACTAGCCTAGCCACCCGATCAGCTCC TGCACTGACTAGCCTAGCCACCCGATCAGCTCC CCGATCGATCGGGCATCGATCGATCGG CCGATCGATCGGGCATCGATCGATCGG CCGATCGATCGGGCATCGATCGATCGG CTAGCCACCCGATCAGCTCCGATCGATCGGG CTAGCCACCCGATCAGCTCCGATCGATCGGG CTAGCCACCCGATCAGCTCCGATCGATCGGG CTAGCCTAGCCACCCGATCAGCTCCGATC CTAGCCTAGCCACCCGATCAGCTCCGATC CTAGCCTAGCCACCCGATCAGCTCCGATC Clustering of fragments based on similar sequences to create contiguous sequence GCCAATGATGCACTGACTAGCCTAGCCACCC GCCAATGATGCACTGACTAGCCTAGCCACCC GCCAATGATGCACTGACTAGCCTAGCCACCC TGCACTGACTAGCCTAGCCACCCGATCAGCTCC TGCACTGACTAGCCTAGCCACCCGATCAGCTCC TGCACTGACTAGCCTAGCCACCCGATCAGCTCC CTAGCCTAGCCACCCGATCAGCTCCGATC CTAGCCTAGCCACCCGATCAGCTCCGATC CTAGCCTAGCCACCCGATCAGCTCCGATC CTAGCCACCCGATCAGCTCCGATCGATCGGG CTAGCCACCCGATCAGCTCCGATCGATCGGG CTAGCCACCCGATCAGCTCCGATCGATCGGG CCGATCGATCGGGCATCGATCGATCGG CCGATCGATCGGGCATCGATCGATCGG CCGATCGATCGGGCATCGATCGATCGG Phasing Analysis Step1: Identify true polymorphic sites Ratio between major and minor alleles needs be above set threshold to be considered as true polymorphic sites The polymorphic sites are determined by a statistical model 5x T 6x G 5x G 5x C 6x A 6x A 10x T 1 x G 5x C 1 x G 10x A 6x A All 3 sites are true polymorphic sites G = noise True Polymorphic site G = noise Build Phase Resolved Contigs Step1: Identify polymorphic sites Step2: Determine which polymorphisms are linked together to resolve two contigs Polymorphic Sites T/G G/A C/A CCATGTTCCAATGATGCCCTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTTCCAATGATGCCCTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTTCCAATGATGCCCTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTTCCAATGATGCCCTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTTCCAATGATGCCCTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG T-G-C are linked CCATGTTCCAATGATGCCCTGTGCATGCATCG CCATGTTCCAATGATGCCCTGTGCATGCATCG CCATGTTCCAATGATGCCCTGTGCATGCATCG CCATGTTCCAATGATGCCCTGTGCATGCATCG CCATGTTCCAATGATGCCCTGTGCATGCATCG G-A-A are linked CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG CCATGTGCCAATAATGCACTGTGCATGCATCG 15
16 Best Matching Alleles Dynamic Phasing Calling polymorphisms from de novo assembled, mapped, paired-end sequences Phase Resolved Consensus Build phased contig sequences based on polymorphic linkage Consensus Alignment Compare contig sequences back to the database to find the best match Best Matching Alleles Build Phased Contig Blocks Detail Review window can be used for in-depth review of HLA genotyping Detail Review window displays the contigalignment browser as well as other reference parameters (ereads and xreads) Contig alignment browser indicates phased blocks Summary Broad coverage (exons & introns) and deep sequencing (> 50) Paired-end sequencing Mapping Phasing: Complement logic (cdna vs. genomic) Paired-end sequencing Central read logic Build Contig blocks 48 16
17 Coverage variance Coverage HLA-A Coverage HLA-B Coverage Position HLA-C Coverage Position 1000 HLA-DQA Coverage Position Position HLA-DQB1 HLA-DRB Coverage Position Position 49 Genotype calling Mapping Reads mapped onto the IMGT-HLA references, including non-classic HLA genes and pseduogenes with NCBI BLASTN. A L R L/R<=2 or R/L<=2 01:02:01 C 01:02:02 01:03:01 Filtering Filtering alignments of sub-best bits, containing mismatches or gaps, and shorter than 50bp, and those where references are mapped to only one end of a pair-end read while one reference is mapped to both ends of the pair-end read, subsequently. B D Genotyping Computing MCOR, MCCR for each mapped reference. Eliminating those of either MCOR = 0 or MCCR = 0. Enumerate combinations of either one reference (homozygous) or two references (heterozygous), and pick up combination of maximum reads 50 Data Analysis Determination of number of reads Bar codes specific for sample and locus (amplicon) Barcodes specific for sample (early pooling) Informatics: Mapping of Reads Phasing Reads Insertions and Deletions Homozygous and Heterozygous Positions Reads from other Loci Hybrid alleles, Novel alleles 17
18 NGS HLA TYPING SYSTEMS 7. Data analysis 1. Sample Collection 5 UTR UTR HLA-A 5 UTR UTR HLA-B 6. Sequencing 5 UTR UTR 5 UTR UTR 5 UTR UTR HLA-C HLA-DQA1 HLA-DQB1 2. Long-Range PCR 5 UTR UTR HLA-DPB1 5 UTR UTR HLA-DPA1 5 UTR UTR HLA-DRB1, 3, 4, 5 5. Library preparation & Pooling 4. Fragmentation 3. Quantification & Pooling Data Analysis Determination of number of reads Bar codes specific for sample and locus (amplicon) - Technically unwieldy - Easier interpretation by Software (reads are assigned to the locus) Barcodes specific for sample (early pooling) - Technically simple - Software needs to be more sophisticated need to phase longer sequence stretches Alleles at different HLA-DRB loci share nucleotide sequences Importance of determining Phase AA Codon DRB1*01:01:01 CA CGT TTC TTG TGG CAG CTT AAG TTT GAA TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGG TTG CTG GAA AGA DRB1*03:01:01: GA- T-C TC- -C- -C- --G AC C --- DRB1*13:01:01: GA- T-C TC- -C- -C- --G C C --- DRB1*13: GA- -T C- --G C C --- DRB3*01:01:02: GA- -T- -G C- --G AC C --- DRB3*02:02:01: GA- -T C- --G C G --- AA Codon DRB1*01:01:01 TGC ATC TAT AAC CAA GAG GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GAG TAC CGG GCG GTG ACG GAG CTG GGG DRB1*03:01:01:02 -A- T-- C G AA T DRB1*13:01:01:01 -A- T-- C G AA T DRB1*13:67 -A- T-- C G AA T DRB3*01:01:02:01 -A- T-- C G T- C DRB3*02:02:01:02 CA- T-- C G A- -C G AA Codon DRB1*01:01:01 CGG CCT GAT GCC GAG TAC TGG AAC AGC CAG AAG GAC CTC CTG GAG CAG AGG CGG GCC GCG GTG GAC ACC TAC TGC DRB1*03:01:01: A G- CG AT DRB1*13:01:01: A A G-C GA DRB1*13: A A G-C GA DRB3*01:01:02: TC C A G- CG AT DRB3*02:02:01: A G- CA AT AA Codon DRB1*01:01:01 AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA GTG CAG CGG CGA G DRB1*03:01:01: TG DRB1*13:01:01: TG DRB1*13: DRB3*01:01:02: DRB3*02:02:01:
19 Informatics: Data Analysis Mapping of Reads Phasing Reads Reads from other Loci (Highly homologous genes, DQA2, DPA2, DQB2, DPB2, DRB2/6/7/8/9) Alleles with incomplete references (in general rare) Hybrid alleles Novel alleles Pseudogene Disambiguation SBT Result: A*02:01 NGS Result HLA-H (novel) A*02:01 TAC CAC CAG TAC GCC TAC GAC GGC AAG GAT TAC ATC GCC CTG AAA GAG GAC CTG CGC TCT TGG H*01:01 GAC CAC CAG TAC GCC TAC GAC AGC AAG GAT TAC ATC GCT CTG AAA GAG GAC CTG CGC TCC TGG (Alpha sample SBC060) Hybrid allele carrying sequences of two loci AA Codon DRB1*01:01:01 GGG C CA CAG TGT GGG CTG GCT TTG GCT GAC ACC CGA CGT TTC TTG TGG CTT AAG TTT GAA CAT TTC TTC AAT ACG A GA- T-C TC- -C- -C- --G DRB1*14:54:01 DRB1*14: A GA- -T C- --G DRB3*02:02:01: C GA- -T C- --G AA Codon DRB1*01:01:01 CGG TGC GAG TTC GGG GAG CGG GTG TTG CTG GAA AGA ATC TAT AAC CAA GAG TCC GTG CGC GAC AGC GAC GTG GAG C C --- -A- T-- C G T DRB1*14:54:01 DRB1*14: C G --- CA- T-- C G A- -C DRB3*02:02:01: C G --- CA- T-- C G A- -C AA Codon DRB1*01:01:01 TAC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAT GCC GAG TAC TGG AAC AGC CAG AAG GAC CTC CTG GAG CAG AGG DRB1*14:54: C- --G --- C G- --- DRB1*14: G A- AA Codon DRB1*01:01:01 CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA GTG CAG CGG CGA G TT GAG DRB1*14:54: A T TG C C-T DRB1*14: G- CA AT C C-T DRB3*02:02:01: G- CA AT C C-T AA Codon DRB1*01:01:01 ACT AAG CAG CTG AGT CCT AAG GTG GTG TAT CCT TCA ACC CAG CCC CTG CAC CAC AAC CTC GTC TGC TCT GTG GGT G T DRB1*14:54:01 DRB1*14: G T DRB3*02:02:01: G --- AA Codon DRB1*01:01:01 GGC AGG TGG GGC GCT ACA TTC TAT CCA AGC ATT GAA GTC TTC CGG AAC CAG GAA GAG AAG GGG GTG GTG TCC GGC T A DRB1*14:54:01 DRB1*14: T A DRB3*02:02:01: C A
20 Characterization of a rare allele with incomplete sequence B*15:147 derives from B*15:01:01:01 20
21 SBT/SSO vs NGS Identifying a novel allele S-101, Reference Type Result: B*13@, B*38@, one allele is an exon 4 variant? NGS Result: B*13:02:01, B*38:02:01 _Exon 4 variant A to G, Lys to Arg, codon 186. DPB1 Hybrid Alleles DPB1*04:02:01:01/DP A1*01:03:01:05 DPB1*463:01/ DPA1*01:03:01:05 E2 X(AAGG) E3 E bpfrom exon2 Recombination area I2 I3 DPB1*03:01:01/ DPA1*01:03:01:03 21
22 Characterization of a Novel allele through the evaluation of unmapped reads Functional Significance Subject with two closely related alleles included in the DPB1*04:02:01:01G DPB1*04:02:01G: DPB1*04:02:01:01 DPB1*04:02:01:02 DPB1*105:01 DPB1*463:01 DPB1*571:01 Identical Antigen Recognition Site Structure Different levels of Expression (we propose) AA Codon DPB1*105:01 CAG CCC CTG ATG TCT ATG ATG GTT CTG GTT TCT GCG GCC CGG ACA GTG GCT ACG GCG TTA CTG GTG CTG CTC ACA *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** DPB1*414:01 DPB1*463: AA Codon DPB1*105:01 AGG G AG CAG TGC GGG GTG GTC CAG GGC GCC ACT CCA AAT TAC CTT TTC GGA CGG CAG GAA TAC GCG TTT AAT ACA *** *** *** *** *** *** *** *** * DPB1*414:01 DPB1*463: AA Codon DPB1*105:01 CTG TAC AAC TTC AGC TTC CAG CGC TTC GAG AGA TAC ATC CGG GAG GAG GTG CGC TTC GAC GAC GTG GGG GAG CGG DPB1*414:01 DPB1*463: AA Codon DPB1*105:01 GAG GAT GAG AAC ATC CGG GCG GTG ACG CTG GGG CGG CCT GAG TAC TGG AGC CAG AAG GAC CTG GAG GAG AAG GCA G DPB1*414:01 DPB1*463: AA Codon DPB1*105:01 GTG CCG GAC AGG ATG TGC AGA CAC AAC TAC GAG CTG GGC GGG CCC ATG ACC CTG CAG CGC CGA G TC CAG CCT AGG DPB1*414: A- DPB1*463: A- AA Codon DPB1*105:01 TCC GGG CCC CAC TGC TTC GTG AAT GTT CCC TCC AAG AAG TTG CAG CAC AAC CTG CTT GTC CAC GTG ACG GAT TAC C C A DPB1*414:01 DPB1*463: C C A AA Codon DPB1*105:01 ATT TTC CTG GAG GTC CTG CCA GGC AGC CAA GTC CGA TGG AAT GGA CAG GAA ACA GCT GGG GTG TCC ACC AAC ATC DPB1*414:01 DPB1*463: AA Codon DPB1*105:01 GAC ATC CTG GAA CAG ACC CGT AAT GGA TGG ACC TTC CAG GTG ATG CTG ATG ACC CCC CAG GGA GAT GTC TAC TGC C T- --- DPB1*414:01 DPB1*463: C T- --- AA Codon DPB1*105:01 CAC AGT CCT GAG TCT AGT CAA GTG GAG ACC AGC CTG GAT GTC ACC GTG TGG A AG GCA CAG GAT TCT GCC CGG AAG C DPB1*414:01 DPB1*463: C AA Codon DPB1*105:01 GGA GTG CTG ATC ATC AGG ACA TTG ACG GCT GGG GGC TTC GGG CTC ATC TGT GGA GTG GGC TTC ATG CAC AGG AGC A DPB1*414:01 DPB1*463:
23 23
24 DPB1 Intron 2 estr(aagg) E1 101 E2 264 X(AAGG) E3 282 E4 111 E UTR F_DPB1 I bp DPB1 Fragment E2/E4 (~5.1kb) I3 R_DPB1 3 UTR Possible estr proximal to intron-2 splicing site STR length may play a regulatory role in the expression of DPB1 DPB1*02:01:04e1 L_AA DPB1*02:01:02v5 L_AA DPB1*02:01:02v6 L_AA DPB1*02:01:02 L_AA Intron2 (-43) Low DPB1*02:01:02v3 L_AA DPB1*02:01:02v7 L_AA DPB1*02:02 L_AA DPB1*02:01:02v2 L_AA DPB1*02:01:02v1 L_AA DPB1*02:01:02v4 L_AA DPB1*04:02:01:01 L_AA DPB1*04:02:01:02 L_AA DPB1*04:01:01:01v1 L_AA DPB1*04:01:31 L_AA DPB1*04:01:31 L_AA DPB1*04:01:01:01v4 L_AA DPB1*04:01:01:01v5 L_AA DPB1*04:01:01:01 L_AA DPB1*04:01:01:01v3 L_AA DPB1*464:01 DPB1*04:01:01:02 L_AA DPB1*398:01 DPB1*30:01 DPB1*58:01e1 DPB1*17:01e1 L_AA DPB1*17:01x1 DPB1*19:01e1 H_GA DPB1*39:01x1 DPB1*11:01:01e1 H_GA DPB1*27:01e1 DPB1*13:01:01/DPB1*107:01e1 H_GA DPB1*85:01e1 H_GA DPB1*01:01:01e1 H_GA DPB1*296:01e1 DPB1*15:01:01e1 H_GA DPB1*18:01e1 H_GA DPB1*05:01:01e1 H_GA DPB1*414:01e1 High DPB1*463:01 DPB1*16:01:01 H_GG DPB1*21:01e1 DPB1*06:01e1 H_GG DPB1*09:01:01e1 H_GG DPB1*104:01e1 DPB1*03:01:01 H_GG DPB1*14:01:01e1 H_GG STR Analysis : Short -Short DPB1* 01:01:01e1, 05:01:01e1 24
25 STR Analysis : Short -Long DPB1* 02:01:02, 13:01:01e1 STR Analysis : Long -Long DPB1* 02:01:02, 02:01:02v3 Data Analysis Determination of number of reads Bar codes specific for sample and locus (amplicon) Barcodes specific for sample (early pooling) Informatics: Mapping of Reads Phasing Reads Insertions and Deletions Homozygous and Heterozygous Positions Reads from other Loci Hybrid alleles, Novel alleles 25
26 Typing two DRB5 alleles All reads need to be accounted Correct genotype: DRB5*01:01:01, DRB5*01:08N DRB5*01:02, 0108N DRB5*01:01:01, 01:02 DRB5*01:01:01, 01:08N DRB5*01:02/01:08N identical in exon 2, differ by 19 nt indel in exon 3 DRB5*01:01:01/01:02 identical in exon 3, differ by 3 nt substitutions in exon 2 Must Know Amplicon: size (homogeneous or variable according to allele families) Preferential amplifications (locus or allele families) Primers: multiplexed or single location Other genes co-amplified (DRB) Software: Binning of reads (to a given locus, to a given allele family). No binning (possible interference in allele assignment) Phasing: reads covering informative SNPs, Central Reads, Assembly Utilization of reads Homozygous allele? Not exactly DRB1 DQA1 DQB1 DRB1 DQA1 DQB1 Count *13:02:01 *01:02:01:04 *06:04:01/*06:09:01 21/553 *15:01:01:01 *01:02:01:03 *06:02:01/*06:03:01 423/553 26
27 DRB1 ~40Kb DQA1 ~10Kb DQB1 DRB1 DQA1 DQB1 *01:01:01/*01:03 *01:01:01 *05:01:01:0x *10:01:01/*14:54:01 *01:05/*01:04:01:01 *05:01:01:02 *01:02:01 *01:01:02 *05:01:01:01 DQB1*05:01:01:0x =DQB1*05:01:01:01(intron 4) +DQB1*05:01:01:02 (intron 2) My thought Process for Genotype Assignment Examine Genotype assigned by software through mapping Perfect match with reference vs no full match at genomic level Check match with reference vs no full match at exon level Check completeness of reference Identify novel allele; see close allele and check differences and sequences Examine by other method phasing (central reads, pair end reads, assembly) Check LD tables (my help identify drop outs) Barcode performance Read count 1.8e e e e+06 1e GTCATCT GTATAGT GTAGCTT GCTGCAT GCGTATT GCATGAT GCAGACT TATACTA TAGTCTC TAGTACG TAGCTAT TACGTCT TACACAT GTGCGAT GTGACGT TATGCGT TATCTGC Barcode 81 27
28 Data Analysis Solid and simple logic error is minor Accurate User-friendly interface for reviewing result Fast Less than 2 hours for seq run (12-24 samples) Ability to pick up new allele Stand-alone desktop solution Ability to evaluate genotype assignment by second method Our experience Allele calls were made virtually by the software with no operator evaluation Fourth field data: in most instances no previous information Haplotype associations stronger than expected Several common allele subtypes distinguished at the fourth field Specific allele associations came apparent without any assumptions made These studies show the robustness and comprehensive coverage provided by the typing system Summary of State of the Art NGS for HLA Application to HLA typing is feasible Processes have been optimized Current methods are appropriate for both Registry Typing and small scale quick TAT Extremely accurate and comprehenisve Great developments in the informatics and analysis Completion of sequences of common alleles will be helpful Studies in familes may unravel limitations 84 28
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