16-point HLA Typing with Long Amplicon Analysis v2 Brett Bowman September 14th, 2016

What is 16-point HLA Typing? “Classical” MHC Proteins 3 CD8-presenting complexes “MHC Class I” HLA-A, B, C 1 Gene per AS 3 CD4-presenting complexes “MHC Class II” HLA-DP, DQ, DR 2 Genes Per AS 2x 1x 1x

AGENDA Long Amplicon Analysis (LAAv2) 16-point HLA Sequencing Results Overview Improvements in LAAv2 for Complex HLA Typing 16-point HLA Sequencing Results Experimental Design Typing Results Sequence Quality

Long Amplicon Analysis WorkFlow – overview Subread Filtering Coarse Clustering Waterfall Clustering Fine Phasing Consensus Post-Processing Filters Outputs Subread Filtering Coarse Clustering Waterfall Clustering Fine Phasing Consensus Post-Processing Filters Outputs Subread Filtering Coarse Clustering Waterfall Clustering Fine Phasing Consensus Post-Processing Filters Outputs Sample Locus Allele Clean-Up Report

Long Amplicon Analysis WorkFlow – Coarse & WATERFALL Clustering Subread Filtering Coarse Clustering Waterfall Clustering Fine Phasing Consensus Post-Processing Filters Outputs Filtered Subreads Clustering Subreads Waterfall Subreads Cluster #1 Cluster #2 Cluster #3

Long Amplicon Analysis WorkFlow – Fine Phasing & Consensus Subread Filtering Coarse Clustering Waterfall Clustering Fine Phasing Consensus Post-Processing Filters Outputs

Results Sample Experimental Designs & Results

What is 16-point HLA Typing? HLA Complete Panel Exon sequence only 2-step PCR 9 outer PCR ~45 inner PCR Phasing by inference 3-day turn around ~QV30 Experimental Design Re-use outer primers 3.1~5.8 kb Active-site genomic 7-loci fully phased >QV50

Long Amplicon AnALYSIS – FULL PANEL RESULTS Sequence gLen gType gPctId nMis Indel cLen cType cPctId Type Cluster0_Phase0_NumReads192 3152 A*34:01:01 100 1093 Cluster1_Phase0_NumReads194 3406 B*15:02:01 99.94 2 1045 B*15:21 Cluster2_Phase1_NumReads203 3395 B*40:01:02 Cluster2_Phase0_NumReads184 3460 C*04:03:01 1096 Cluster3_Phase1_NumReads228 3445 C*12:03:01:01 C*12:03:01 Cluster3_Phase0_NumReads215 4839 DPA1*01:03:01:04 96.88 52 601 DPA1*02:02:02 Cluster4_Phase0_NumReads234 5801 DPB1*03:01:01 99.31 39 1 657 DPB1*01:01:01 Cluster5_Phase1_NumReads178 4572 DPB1*04:02:01:02 99.47 18 6 Cluster6_Phase1_NumReads201 5743 DQA1*01:02:01:01 99.97 613 DQA1*01:02:01 Cluster5_Phase0_NumReads240 5692 DQA1*03:03:01 99.95 3 Cluster7_Phase1_NumReads236 3768 DQB1*03:05:01 99.28 19 8 618 DQB1*04:02:01 Cluster6_Phase0_NumReads216 3855 DQB1*05:01:01:01 99.52 16 DQB1*05:02:01 Cluster8_Phase1_NumReads187 5105 DRB1*04:03:01 99.05 17 31 584 DRB1*04:05:01 Cluster7_Phase0_NumReads217 3893 DRB1*15:01:01:03 DRB1*15:02:01 Sequence gLen gType gPctId nMis Indel cLen cType cPctId Type Cluster0_Phase0_NumReads195 3138 A*11:01:01:01 100 1093 A*11:01:01 Cluster1_Phase1_NumReads173 3153 A*74:02:01:02 99.84 5 A*74:01 Cluster1_Phase0_NumReads189 5778 B*07:02:01 1045 Cluster2_Phase1_NumReads179 3398 B*53:01:01 Cluster2_Phase0_NumReads212 3424 C*04:01:01:01 1096 C*04:01:01 Cluster3_Phase1_NumReads222 3429 C*07:02:01:03 C*07:02:01 Cluster4_Phase1_NumReads188 4830 DPA1*01:03:01:02 97.06 103 40 601 DPA1*02:01:01 Cluster3_Phase0_NumReads230 4857 DPA1*01:03:01:04 DPA1*01:03:01 Cluster5_Phase3_NumReads200 4574 DPB1*02:01:02 99.58 18 1 107 DPB1*01:01:01 Cluster4_Phase0_NumReads186 5800 DPB1*03:01:01 99.48 28 2 657 DPB1*107:01 Cluster5_Phase2_NumReads225 4569 DPB1*04:01:01:01 99.98 Cluster5_Phase1_NumReads220 5872 99.86 8 DPB1*04:01:01 Cluster5_Phase0_NumReads201 5751 DQA1*01:02:01:04 99.93 3 613 DQA1*01:02:01 Cluster6_Phase1_NumReads228 5691 DQA1*03:03:01 Sequence gLen gType gPctId nMis Indel cLen cType cPctId Type Cluster0_Phase0_NumReads176 3138 A*01:01:01:01 100 1093 A*01:01:01 Cluster1_Phase1_NumReads177 3152 A*02:01:01:01 A*02:01:01 Cluster1_Phase0_NumReads228 3395 B*08:01:01 1045 Cluster2_Phase1_NumReads189 B*40:01:02 Cluster2_Phase0_NumReads171 3422 C*03:04:01:01 1096 C*03:04:01 Cluster3_Phase1_NumReads200 3430 C*07:01:01:01 C*07:01:01 Cluster3_Phase0_NumReads175 4858 DPA1*01:03:01:02 602 DPA1*01:03:01 Cluster4_Phase0_NumReads193 4568 DPB1*04:01:01:01 DPB1*01:01:01 Cluster5_Phase1_NumReads210 5875 99.95 1 2 657 DPB1*04:01:01 Cluster6_Phase1_NumReads210 5691 DQA1*03:01:01 613 Cluster5_Phase0_NumReads235 5799 DQA1*05:01:01:02 610 DQA1*05:01:01 Cluster6_Phase0_NumReads239 4114 DQB1*02:01:01 99.98 618 Cluster7_Phase1_NumReads210 3775 DQB1*03:02:01 99.92 Cluster7_Phase0_NumReads175 3686 DRB1*03:01:01:01 584 DRB1*03:01:01

Long Amplicon AnALYSIS – Class I Results HLA Class I 3 genes 3.1~3.4 kb gDNA ~1.5 kb active-site 3 Sanger rxns ea ~QV30 Experimental Design 45x samples All Class I genes Complete genomic Fully phased >QV50

Introducing – Loci Analysis LociAnalysis -vv -n 16 -o OUTPUT_DIR --combineLoci DPB:DPB1:DPB2 --ignoreLoci DPB1,DPB2,H --minLengthByLocus DRB4:900 --maxLengthByLocus DRB4:2000 --maxClusteringReadsByLocus DRB1:500 --maxReadsByLocus DRB1:3000 DATABASE_FOLDER SUBREADS.BAM