Computational methods for genomics-guided immunotherapy Sahar Al Seesi

Computational methods for genomics-guided immunotherapy Sahar Al Seesi

Computational methods for genomics-guided immunotherapy Sahar Al Seesi and Ion Mndoiu Computer Science & Engineering Department University of Connecticut Class I endogenous antigen presentation Somatic rearrangement of T-cell receptor genes Potential TCR repertoire diversity: 1015 T-cell selection in thymus Estimated TCR repertoire diversity after selection: ~2x107 T-cell activation and proliferation T-cell activation and proliferation T-cell activation and proliferation The immune system and cancer Cutting the brakes: PD1 and CTLA-4 blockade Stepping on the gas: vaccination with neoepitopes Combined approach Ton N. Schumacher, and Robert D. Schreiber Science 2015;348:69-74 Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Sequencing QC and Mapping Tumor DNA Calling SNVs Epitope Prediction Normal DNA Clonality Analysis Vaccine Design Tumor RNA Whole Genome or Nextera Rapid Library prep Capture Exome Whole Transcriptome Library prep Illumina HiSeq Sequencing TCR Sequencing Sequencing Tumor DNA QC and Mapping Calling SNVs Normal DNA Whole Genome or Exome Library prep AmpliSeq Ion Proton Sequencing Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Tumor RNA Whole Transcriptome Library prep Ion PGM Sequencing QC and Mapping Sequencing Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing ION-Torrent Proton Runs: read statistics # of reads # of bases

Mean of reads lengths std of reads lengths PB 25,031,340 3,272,787,408 130.74 66.88 Melanoma 1T Patient 2T 19,252,932 2,589,624,915 134.5 68.67 28,400,728 4,147,914,801 146.04 66.91 3T 26,039,006 3,800,446,471 145.95 67.02 Normal 20,726,352 3,353,732,704 161.81 63.35 20,726,352 3,360,840,809 162.15 63.14 20,726,352 3,367,827,877 162.49 62.92 Synthetic TumorAF10 Tumor TumorAF20 Sequencing QC and Mapping Calling SNVs Epitope Prediction Tumor Exome Reads Human reference Normal Exome Reads Human reference Tumor RNA-Seq Reads Clonality Analysis Vaccine Design TCR Sequencing Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis fastq QC Tools Tools to analyze and preprocess fastq files FASTX (http://hannonlab.cshl.edu/fastx_toolkit/) Charts quality statistics Filters sequences based on quality Trims sequences based on quality Collapses identical sequences into a single sequence Vaccine Design TCR Sequencing Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing fastq QC Tools

Tools to analyze and preprocess fastq files PRINSEQ (http://prinseq.sourceforge.net/) Generates read length and quality statistics Filters reads based on length, quality, GC content and other criteria Trims reads based on length/position or quality scores Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design Mapping decisions What is the best mapper for your data? TCR Sequencing Tumor Exome Reads Human reference End-to-end unspliced alignments vs. spliced or local alignments Normal Exome Reads Human reference Unique vs. non-unique alignments Tumor RNA-Seq Reads Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing ION-Torrent Proton read mapping comparison Bowtie2 Melanoma Patient Synthetic Tumor TMAP Segemehl % of aligned bases Aligned reads length mean % of aligned bases Aligned reads length mean % of aligned bases Aligned reads 2 length mean2 PB 89% 138.8 100% 130.7 99% 135.4 1T 90% 143.3 100% 134.5 99% 140.4 2T 90% 153.3 100% 146 99% 150.3 3T 91% 153.4 100% 146 99% 150.4 Normal 89%

172.89 100% 161.81 98% 167.66 Tumor_AF10 89% 173.03 100% 162.15 99% 167.87 Tumor_AF20 89% 173.17 100% 162.49 99% 168.08 Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Tumor Exome Reads * Human reference * * * * * * * * * Normal Exome Reads Vaccine Design TCR Sequencing Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design Tumor Exome Reads Somatic Variant Callers Mutect (Broad Inst.) VarScan2 (Wash. U.) SomaticSniper (Wash. U) Strelka (Illumina) SNVQ w/ subtraction (UConn) TCR Sequencing * Human reference * * * * * * * * * Normal Exome Reads QC and Mapping Sequencing Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Coverage distribution of exome vs. SNV calls 40% 35%

30% % of variants called 25% CCDS HCC1954BL CCDS HCC1954 Mutect SNVQ Sniper Strelka Varscan2 20% 15% 10% 5% 0% 0 20 40 60 80 100 Read depth 120 140 160 180 200 Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Comparing Somatic Variant Callers Synthetic Tumors ION Torrent Proton exome sequencing of two 1K Genomes individual (mutations known) Downloaded from the public Torrent server Both exomes were sequenced on the same Proton chip Subset of the NA19240 sample was used as the normal sample Mixtures of NA19240 and NA12878 samples were used as the tumor samples Reads were mixed in different proportions to simulate allelic fractions, 0.1, 0.2, 0.3, 0.4 and 0.5. QC and Mapping Sequencing Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Comparing Somatic Variant Callers mixture_AF_10 mixture_AF_20 mixture_AF_30 mixture_AF_40 mixture_AF_50 TP FP FN TP FP FN TP FP FN TP FP FN TP FP FN Bowtie2 464 1,232 11,823 2,506 1,224 9,781 4,608 1,219 7,679 6,138 1,259 6,149 7,018 1,322 5,269 SNVQ Strelka Mutect Sniper Varscan2 TMAP Semegehl Bowtie 2 TMAP Semegehl Bowtie 2 TMAP Semegehl Bowtie 2 TMAP Semegehl Bowtie 2 TMAP Semegehl 476 470 1,573 1,738 402

440 360 22 10 17 92 91 7 341 295 331 218 85 80 80 3,050 1,469 1,344 1,971 1,056 20 11,811 11,817 10,714 10,549 11,885 11,847 11,927 12,265 12,277 12,270 12,195 12,196 12,280 2,605 2,668 4,220 4,475 1,094 1,183 994 35 15 20 797 836 837 341 286 302 191 55 54 54 3,178 1,548 1,422 1,978 1,015 447 9,682 9,619 8,067 7,812 11,193 11,104 11,293 12,252 12,272 12,267 11,490 11,451 11,450 4,842 4,910 5,776 6,089 1,526 1,637 1,379 61 31 29 1,922 1,994 1,994 364 307 317 199 43 39 46 3,235 1,618 1,471 1,952 1,013 476 7,445 7,377 6,511 6,198 10,761 10,650 10,908 12,226 12,256 12,258 10,365 10,293 10,293 6,404 6,465 6,660 7,005 1,797 1,907 1,614 89 48 44 3,198 3,298 3,312 378 333 362 241 40 40 46 3,338 1,661 1,528 2,018 1,071 505 5,883 5,822 5,627 5,282 10,490 10,380 10,673 12,198 12,239 12,243 9,089 8,989 8,975 7,309 7,359 7,232 7,592 1,937 2,037 1,732 163 104 41 4,284 4,466 4,489 394 357 388 286 41 49 49 3,375 1,763 558 2,162 1,161

590 4,978 4,928 5,055 4,695 10,350 10,250 10,555 12,124 12,183 12,246 8,003 7,821 7,798 QC and Mapping Sequencing Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Comparing Somatic Variant Callers 120 100 PPV 80 Strelka SNVQ Mutect Sniper Varscan2 60 40 20 0 0 10 20 30 40 Sensitivity 50 60 70 Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing The ICGC-TCGA DREAM Somatic Mutation Calling Challenge Initial Goal: Find the Best WGS Analysis Methods Challenge 1 Data: 10 Real Tumor/Normal pairs 5 from pancreatic tumors and 5 from prostate tumors Sequenced to ~50x/30x Up to 10K candidates will be validated re-sequencing to ~300x coverage using AmpliSeq primers on an IonTorrent Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Criteria for selecting candidate epitopes 1) Gene harboring the SNV must be expressed (FPKM estimation) IsoEM (Nicolae et. al., Algorithms for Molecular Biology, 2011) http://dna.engr.uconn.edu/?page_id=105 RSEM (Li et. al., BMC Bioinformatics, 2011) http://deweylab.biostat.wisc.edu/rsem/ Not Expressed X Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing

Criteria for selecting candidate epitopes 1) Gene harboring the SNV must be expressed 2) Peptide will be generated inside the cell upon protein being cleaved by the proteasome 3) Peptide will bind to an MHC molecule that will chaperon it to the cell surface NetChop Predicts cleavage sites of the human proteasome http://www.cbs.dtu.dk/services/NetChop/ SYFPEITHI Predicts MHC I, MHC II binding http://www.syfpeithi.de/ NETMHC Predicts MHC I binding http://www.cbs.dtu.dk/services/NetMHC/ NetCTL Combined cleavage and MHC biding predictions http://www.cbs.dtu.dk/services/NetCTL/ Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Candidate neo-epitopes statistics for two mouse cell lines Duan et. al., JEM 2014 Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design Epi-Seq pipeline for neo-epitope prediction on local Galaxy server TCR Sequencing Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Rational vaccine design requires info on the clonal structure of the tumor Not all cells harbor all candidate epitopes Approaches to clonality analysis 1) Computational inference from sequencing depth SNV allelic fractions only 2) Targeted amplicon sequencing of selected mutations at single cell level More noisy data, potentially biased by capture protocols Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design Cell capture & pre-amp TCR Sequencing Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design PCR on Access Array TCR Sequencing Sequencing QC and Mapping Calling SNVs Epitope

Prediction Clonality Analysis Vaccine Design PCR on Access Array TCR Sequencing QC and Mapping Sequencing Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Captured cells in pilot run 1 2 3 4 5 6 7 8 9 10 11 12 A 1_C03 1_C02 1_C01 1_C49 1_C50 1_C51 1_C06 3_C05 1_C04 1_C52 1_C53 1_C54 B 2_C09 1_C08 1_C07 1_C55 1_C56 1_C57 1_C12 1_C11 1_C10 1_C58 1_C59 1_C60 C 1_C15 2_C14 2_C13 1_C61 1_C62 1_C63 1_C18 2_C17 1_C16 1_C64 1_C65 1_C66 D 1_C21 2_C20 1_C19 1_C67 1_C68 1_C69 2_C24 2_C23 4_C22

1_C70 1_C71 1_C72 E bulk 2_C26 1_C27 1_C75 1_C74 1_C73 0_C28 0_C29 0_C30 1_C78 1_C77 2_C76 F 1_C31 0_C32 1_C33 1_C81 1_C80 1_C79 0_C34 1_C35 0_C36 1_C84 0_C83 1_C82 G 0_C37 1_C38 0_C39 1_C87 1_C86 1_C85 1_C40 1_C41 1_C42 1_C90 1_C89 1_C88 H 1_C43 1_C44 1_C45 1_C93 1_C92 1_C91 1_C46 1_C47 1_C48 1_C96 1_C95 1_C94 Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Analysis Pipeline Barcode list Fastx Barcode Splitter 96 fastq files: one per well pooled fastq file tmap mm9 BALBc genome Generate Referece List of SNV Locations 96 sam files: one per well fasta with +/300 bases around each

SNV compute coverage 96x48 with total and fwd/rev variant coverage for each well/SNV Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Target Aligned Reads 180,000 160,000 140,000 120,000 100,000 80,000 60,000 40,000 20,000 - 180,000 160,000 140,000 120,000 100,000 80,000 60,000 40,000 20,000 - Unaligned Reverse Unaligned Forward Reverse Forward QC and Mapping Sequencing Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Per SNV Coverage 250000 200000 150000 100000 alt_rev alt_fwd ref_rev ref_fwd 50000 0 20 0: 0 5 59 r1 1 ch chr 82 10 1: 4 13 24 8 96 32 0 75 45 1 83 74 7 84 72 4 54 30 8 63 49 1 22 68 7

17 74 6 45 75 1 75 96 4 71 86 8 76 42 3 82 06 5 83 56 9 57 15 3 27 67 7 51 35 6 3 8 5 4 2 7 6 1 2 7 0 0 8 2 2 9 1 :6 57 26 9 1 1 14 73 55 85 27 53 24 10 4 16 6 14 6 62 17 71 30 1 : : : : : : : : : : : : 1 r1 r4 hr6 hr6 hr7 hr8 1: 2: 2: 3: 1 2 r 1 3 hr 1 r 1 5 r 1 7 r 1 7 r 1 9 r 2 : ch chr hr1 c ch c c c c hr h h h h h h hr hr c c c c c c c c c c 16 8 79 28 SNVs QC and Mapping Sequencing Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR

Sequencing SNV Support Matrix Cells A1 Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse Alt in forward Alt in reverse 0 0 0 2 0 4 0 1 - - - A2 - A3 0 0 000 0 2 3 01- - - 016 0 0 0 1001- - A4 1239 3189 0 3 3 5 1408 2390 1 1 0 1 1 1 - 1 2 0 1 1 0 0 0

- 1634 2536 0 1 0 0 0 0 00- 1060 2638 435 2760 945 212 7 5 3 3 - 0 0 A5 0 7 1 0 0 5 0 6 462 638 0 1 351 1284 1 3 2 5 1852 1974 0 0 1361 1440 0 2 0 0 1 5 1520 1005 1348 1581 1982 2282 1207 1405 0 0 0 0 1 2 0 0 0 0 0 4 0 2 0 0 0 0 0 0 0 0 0 5 9 6 0 4 656 818 0 1 2 0 337 80 0 3 0 0 3 3 A6 0 1 652651 4018 291 471 - 889 1342 0 2 0 0 708 1035 0 0 1 6 0 0 1757 2340 0 1 0 1 0 0 0 3 1 0 0 0 1 0 0 6 - 25 50

47 79 1367 1183 1388 1110 2340 1146 332 207 0 0 - 0 0 0 1 0 1 815 153 - 8 6 0 0 1175 1591 1 0 174 218 0 0 873 1102 606 1215 0 1 307 1212 1742 1590 0 0 0 1 627 1057 0 0 - - A8_3 1 2 103 144 338 545 262 442 338 394 164 539 1 0 170 238 301 394 0 0 906 1085 680 833 521 757 1225 1229 225 238 47 46 0 0 - - A7 1892 1312 - 0 0 00183 109 1082 890 0 0 001177 3 2140 2188 0 0 1902 1629 9 14 0 0 1646 1748 18 8 1956 1433 0 0 84 124 0 0 00- A9 610 759 11 12 688 1216 258 382 1 0 173 1097

2 1 1201 1692 4 6 1124 717 142 180 510 742 0 0 2 2 375 228 759 1292 0 0 15 19 708 1387 18 21 332 1227 0 0 1 0 1 0 550 596 1 2 266 380 37 83 558 580 325 785 3 2 1 1 0 1 0 0 966 940 0 0 974 1259 24 27 310 407 286 678 375 88 991 241 330 550 237 211 0 1 0 0 1227 2073 0 0 5 1 0 0 1115 1377 221119 1135 0 0 - 0 1 - 182 232 84 56 1342 1709 0 0 1 0 332 733 00682 803 - 0 0 605 732 2 3 0 0 0 0 1534 2090 3 6 85 218 183 273 1 0 - 2 1 1121 1478 859 1063 0 1 0 4 14 63 0 0 0 0 A11

24 21 1 1 986 1245 520 638 719 678 - 1 0 541 8651 990 A10 - 0 2 - - 52 31 27 11 26 688 1376 1303 2 1 0 0 375 354 12 7 1285 1101 1293 502 1078 867 0 0 0 0 1 1 0 0 0 0 000 0 311 375 0 12 00- 108 152 4 5 1476 2225 675 854 0 0 342 58 0 0 1049 1583 0 0 1 1 A12 0 0 958 759 748 942 1 0 5 9 002 8 307 220 2 6 0 1 600 440 0 139 0 0 11 13 0 0 16 86 0 0 3 2 449 251 0 0 B1_2 0 0 132 117 1345 1964 562 629 0 0 972 657 16 609 850 1050 659 953 136 85 1066 1150

697 532 3 0 342 473 970 458 179 160 0 2 2 5 737 1181 332 660 0 0 788 849 1 3 311 356 1 0 1 1 0 3 798 782 106 103 343 2054 276 1065 1091 754 649 96 215 212 0 0 2 3 B2 509 429 1282 1412 634 1012 678 942 261 358 0 0 11 288 234 313 1011 1471 730 977 745 883 812 939 2 1 1002 1214 209 406 636 253 941 1086 931 1255 766 988 35 48 569 530 176 489 2 0 169 243 131 145 107 165 1 2 0 44 2 3 635 9 1692 1873 7 8 392 1240 563 791 302 1001 1 0 495 16 38 38 0 0 191 245 B3 501 471 1545 1132 0 1 1 0 2450 2307 1 0 293 227 1429 1152 1708 1620 0 0

1059 1388 0 0 0 2 0 1 3 6 0 1 1517 1285 1072 1421 1511 217 584 97 4 5 4 3 B4 101 132 688 832 351 681 213 333 83 111 0 1 104 439 0 1 888 1430 595 758 1 0 859 931 0 0 139 139 532 886 521 428 1122 1604 853 1317 435 585 634 1264 48 117 216 655 60 77 0 1 801 1074 347 468 102 147 0 133 0 0 83 0 518 754 4 6 197 525 845 1182 200 372 563 99 816 192 132 215 16 14 0 4 B5 0 2 0 1 0 2 0 2 0 0 50 2309 0 0 7 12 0 2 0 2 0 2 0 1 0 1 0 4 0 8 0 0 4 4 0 1 0 0 0 1 0 0 0 4 1 2 1

0 0 1 0 0 0 2 0 0 0 1 0 3 0 1 4 4 0 0 0 0 B6 79 98 2 0 3 1 0 2 3 1 773 725 424 1465 1153 1416 6 6 1739 1720 0 0 744 715 0 1 1944 2043 358 522 3 1 26 35 3 5 0 2 3 4 0 0 363 1000 0 0 0 0 2 2 856 903 5 2 0 413 001425 10 1708 2098 4 2 626 1137 1630 1691 477 918 911 159 0 0 1 6 011103 1178 B7 0 1 1345 604 2641 1971 21 16 0 0 1190 482 0 0 638 721 2939 1726 0 0 1166 355 1648 664 134 64 0 4 903 486 0 1 279 147 1903 989 2202 1027 581 512 0 0 351 580 0 2 0 0 0 0 1164 497 798 458 0 89

1628 2 0 0 0 0 2089 1313 0 3 1414 1248 1627 141 0 0 3033 1926 4 2 B8 2 0 529 668 285 519 632 1001 180 288 1 0 227 1142 786 1138 3 4 0 0 233 247 303 422 1508 1979 569 849 0 0 652 1019 0 0 251 344 1 1 178 421 0 1 0 0 281 489 178 282 1 0 3 2 0 148 1 0 0 0 422 536 0 6 201 521 426 547 181 431 1 0 1 0 6 16 220 183 308 369 B9 828 1093 618 826 666 1967 1 0 34 62 0 0 98 672 46 63 834 1245 689 1023 717 438 567 798 489 839 203 315 337 597 210 146 567 1241 0 1 1029 62 1 0 95 391 0 0 5 8 123 185 39 62 23 11

0 234 1 0 788 28 326 444 1 3 553 1818 640 960 158 398 25 28 756 87 11 22 29 25 784 1210 B10 419 461 547 548 1472 1879 143 210 39 47 430 336 79 194 218 518 42 43 126 166 505 336 710 736 574 543 131 135 695 810 260 278 187 189 128 132 B11 00- - 1 0 1 0 2 4 1 0 - 3381 3807 - 5 0 0 0 0 0 238 496 234 449 22 100 582 703 705 934 463 507 313 323 1 4 0 20 455 515 103 0 767 1002 4 3 45 63 658 709 406 597 365 70 533 122 1 1 88 73 0 1 1680 2550 0 1 1 0 0 1 1 0 0 0 0 0 0 0 100 0 1 0

3 5 1 0 B12 1354 1170 20282 327 1094 490 222 0 1 3 1015 547 331 250 10630 370 118 190 001 1 2 1 0002397 429 164 159 000 1 128 165 068 374 401 10260 307 2401123 197 962 104 1262 266 05345 225 1378 1330 - C1 - C2_2 - C3_2 0 1 00- - 7 2 00- 1 0 0 0 3 2 1809 1341 1 0 0 0 - 3188 4677 0 0 0 0 1 4 000 2 000 0 - 0 0 3 7 - 0 1 1528 2488 0 0 2384 2539 0 1 0 1 0 1 1 2 0 0 - 0 0 12 9 10- - 0 1 1 4 - 9 6 0 0 C4 913 1083 1050 1262 299 430 432 673 143 182 10 9 14 564 553 769 842 1197 68 108 512 384 326 405 586 673 1785 2215 302 416 361 233 760 1003 440 577

694 967 506 868 511 738 28 87 1 6 385 672 574 742 507 772 306 359 0 154 0 0 1098 10 904 1272 1 2 173 812 465 584 190 750 574 512 585 136 291 363 144 125 220 277 C5 1668 1992 2172 2345 0 1 0 3 0 1 1799 1949 0 5 1397 1731 4 12 2130 2245 0 1 1996 2160 - 1218 1413 456 428 1059 2553 553 777 41 65 969 980 0 0 0 1 2 1 982 1142 0 0 200 233 0 0 2710 3487 0 0 0 0 139 201 926 1225 65 73 0 0 444 1138 51 163 0 0 1263 1717 0 0 887 1110 1 1 0 308 0 4 0 0 246 355 4 6 309 864 1205 1661 0 3 0 1 5981 1942 0 1 7 4 2 0 1 0

0 0 0 0 0 7 0 3 1984 2662 0 10 1819 2188 0 6 0 1 0 4 0 3 83 128 1953 2140 0 3 3 1 0 1 0 0 C6 2 0 - 0 0 0 0 2 2 1 0 1 1 0 0 0 0 - 0 4 6 18 0 4 0 11 0 5 334 71 27 59 0 0 1 3 2 0 0 0 1 1 1 0 0 1 - C8_2 - 415 645 993 980 354 892 - 4 7 - - - - - - - - - 17 5 - C7 - 920 155 1060 1686 880 714 556 663 - 302 271 44 56 572 626 1128 1453 968 1045 117 179 415 1187 838 1134 0131001003 10 490 704 00675 1206 279 68 2300- C9 0000180 159 101588 2433 555 793 - - - - - - - C10 - C11 0 0

0 3 - 1 7 0 0 80009 0 10000 0 103 3 - - 11- - 0 0 - - 4 8 0 0 C12 248 224 1099 1436 370 518 56 50 163 870 937 994 20619 652 10831 779 1064 1180 1955 2033 111537 1774 381 379 474 411 511 835 00134 145 00832 850 500180 130 140 883 11874 2176 380051 115 1764 298 177 229 0016- D1 D2_2 0295- 000 0 - - 1909 3151 0013 10 - 710 1004 1909 2092 0 0 1051 1323 0 0 2 0 0 1 83 133 1005 1321 - 1178 1591 010 0 0 1 0 0 0 0 001 0 - 1 0 0 0 1 0 0 1 0 0 0 0 0 0 - 1 0 8 9 2 0 10- - 0 1 0 0 0 0 641 989 6 8 - - - - 1 2 D4 1112 1285 1184 1499

2209 3522 57 41 28 933 201 777 490 4 3044 3930 0 0 1563 2184 0 0 D3 1 1 577 889 - - 1977 2623 263 531 2 0 488 677 1041 1344 469 917 0 0 0 1 0 0 0 1 911 1324 26 41 875 855 0 0 0 0 320 6 0 0 4 9 1 1 858 1218 13 59 1363 1290 0 0 1270 1906 4 4 1192 1863 - - - D5 0 1 0 1 0 5 130202004 6 0 9 22110 021 0 04010 2 0 3 1 8 0202100 0 0 4 0 1 0 0 01013200071303107 9 0 0 01- D6 00100 0 - 5 3 00- D7_2 0 0 807 1104 157 362 362 633 83 117 1 0 120 808 0 0 1 4 223 330 400

320 501 725 0 0 718 1026 14 19 0 0 4 9 1 10 3 11 6 3 0 0 1 0 0 0 - 0 0 10- 0 0 10 19 712 2 3 0 0 - 0 0 0 0 0 0 0 1 00- 0 0 0 0 1 0 0 0 000 0 0 1 0 0 6077 1465 3 7 - 3 3 0 0 2 1 3 5 0 0 0 0 D8_2 - 1 0 0 0 4 2 6 6 1 0 0 0 1620 2097 - 311 184 439 879 002 0 38 125 259 895 39 46 1 1 537 682 280 428 852 784 0122 245 2 56 104 0 1 29 89 892 1188 145 319 61 13 650 1248 139 90 0 0 Low D9_4 1762 2102 2 0 0 0 0

1 4 1 1 0 1 2 680 1653 0 0 2 0 1 0 2 0 2 0 0 0 0 0 0 0 0 1 2 3 0 0 5 0 516 728 598 746 780 1240 234 362 133 269 296 370 172 503 172 423 706 1108 341 471 394 287 452 536 327 467 736 1005 282 482 133 217 472 848 200 354 552 805 216 457 193 410 81 288 242 369 194 366 201 241 293 382 201 294 0 81 171 228 702 5 449 791 3 6 497 752 505 703 189 405 376 58 616 167 394 667 186 189 248 355 D10 - D11 0 0 1 0 1 5 0 0 0 0 - 1 0 1 0 0 0 0 0 0 0 0 0 0 0 5 8 -

D12 0 0 935 1101 1325 2478 0 0 371 499 885 741 33 1227 460 725 86 155 723 994 206 140 68 88 23 28 1 0 317 189 1324 1575 405 567 511 642 140 339 0 0 0 0 110 141 1 4 0 0 671 879 22 11 0 76 996 1410 29 1 3 1 0 0 34 268 299 472 56 206 174 174 558 141 973 1306 0 0 108 246 351 266 1213 919 662 589 811 730 279 221 229 134 40 507 43 52 1160 1155 494 486 766 309 563 431 759 514 2068 1741 217 198 492 356 712 471 0 1 28 37 63 166 149 139 0 0 73 72 556 474 509 361 0 4 0 0 882 8 1301 1241 3 2 2 12 844 745 137 313 579 333 781 113 934 805 311 167 173 186

E1_bulk 349 505 362 623 587 1157 318 624 258 452 263 303 25 1021 383 745 664 1327 368 698 448 486 353 488 299 463 749 1389 413 808 320 256 421 786 281 443 475 939 204 549 230 421 98 322 137 247 167 396 414 760 179 354 290 418 0 158 169 287 686 16 284 584 2 3 140 867 479 1060 113 642 362 535 519 214 440 741 136 145 159 424 E2_2 - E3 1486 2574 3 6 1 0 0 0 0 0 0 0 0 1 0 1 2 1 0 2 1 0 837 1151 0 0 0 1 0 0 3548 4377 0 0 1 0 4 7 - E4 000 1 1171 1889 0 0 199 317 466 388 54 159 238 528 1188 1414 701 624 82 55 1134 1206 585 640 1808 2130 262

333 582 586 260 379 452 510 1221 1343 148 277 0 0 251 708 1 2 359 509 408 440 395 408 491 597 0 147 16 25 899 6537 835 2 2 1744 2897 717 889 407 775 930 115 936 232 97 154 60 52 457 481 E5 E6 0 1 396 418 2475 3913 0 1 0 0 1654 1726 0 2 1 2 1333 1834 606 616 0 0 733 841 994 1166 0 4 1019 1465 841 629 146 187 0 1 0 0 847 1315 1317 1863 577 1426 0 2 1 1 1795 2165 1 3 4 2 0 3 168 198 - - 0 1 0 0 4 2 2 0 0 1 0 0 0 0 - 2949 3782 6 6 1358 3125 0 0 0 1 1010 391 0 1 2 6 0 0 5 2 E7_0 0

2 0 1 1784 3342 0 0 002 2 0 0 0 0 - High 0 0 663 769 286 1145 796 1248 2 3 0 0 0 1 1 2 0 2 0 2 0 0 0 1 1377 2195 0 0 1297 1602 0 3 0 3 0 1 0 0 1 3 0 0 0 0 0 2 0 1 0 0 0 0 107 178 1405 3486 0 1 523 1232 0 0 449 115 1 2 0 0 3 2 - - - - E8_0 00014 7 0032111 1 4 2 01000110005 3 250 1 00000000- 0 1 0 0 0 0 0 0 0 0 0 0 3 6 1 3 2 6 0 0 3 0 4 4 0 0 0 0 E9_0 - 0 0 0 1 1 0 1 1 1 0 0

0 2 6 0 0 294 1248 1 0 866 1385 879 939 856 620 1102 1249 270 407 0 1 431 647 1145 810 0 0 1025 1487 0 0 1 0 0 1 0 1 0 0 - 1 1 0 1 2 0 - - 001 0 0 0 0 0 100 0 0 0 01- - 0 0 0 1 0 1 4 8 - 1017 1231 483 441 1734 2872 682 962 0 1 1 0 0 0 - E10 0 0 0 1 0 8 - E11 - E12_2 3 3 0 0 0 0 - 344 550 443 829 0 0 001 0 0 0 20031 454 590 643 1329 453 281121 2557 574 719 542 1188 00470 115 24 36 00530 658 1 0 0 0 0 0 0 0 0

0 2 0 1099 3269 1657 1987 3 3 0 2 2 0 743 912 477 731 425 755 108 182 59 85 50 248 469 680 279 467 511 840 1 0 403 501 73 98 0 0 217 394 166 151 115 175 0 1 712 977 160 344 0 0 116 533 192 342 138 226 451 604 51 81 3 8 0 47 211 267 664 8 1243 1878 0 2 129 285 92 154 196 430 201 52 545 164 405 784 1 0 5 1 F1 - F2_0 2 0 6 8 5 0 0 2 1 1 0 0 1 0 2 1 3 1 2 2 0 0 0 0 0 2 1 0 2 2 0 0 - - - F3 0 1 0 0 00002 0 - 428 711 1292 1417 0 1 1 3 1395

1787 1135 1375 824 1041 227 326 727 833 1606 2370 - 010 0 1595 1994 0 0 17 33 50 186 1091 1854 52 50 - 432 632 0 546 0 0 684 2 1251 1875 7 4 219 451 501 737 0 0 0 0 641 179 1087 1812 0 0 377 484 F5 2389 2727 1 3 1068 1598 0 5 - - 001064- F4 1332 1776 997 1117 0 1 2 9 0 0 0 0 2470 2775 0 4 0 2 1298 1443 0 0 1 2 0 2 0 0 0 3 0 1 0 1 0 135 920 980 0 2 0 0 2 1 391 720 393 1427 - 2 12 - F6 1 4 0 0 1 13 0 0 0 1 0 5 0 2 0 9 5 9 1 9 0 3 0 4

0 5 6779 1154 0 2 0 3 0 5 0 0 0 4 1 0 2593 1696 0 1 0 0 0 1 0 4 0 2 0 1 0 1 0 0 1 0 6668 1263 12 10 1 17 0 6 0 1 - - - - - - 12 6 0 1 0 1 F7_0 10102 0 2 0 102 0 3 3 21001 0 000 0 2 2 002 0 0 0 1771 2801 0 1 001 0 110 1 0 0 0 0 13 62 0 0 0 0 0 7 4 0 0 0 0 F8 4 1 1 0 F9_0 200 0 2 1 0 0 3 0 2 0 4 3 1 1 4 3 1 0 0 0 0 0 2 0 3 0 0 0 0 0 007 1 200 1

0 0 0 0 0 0 0 0 0 0 5 0 0 0 00- 4 4 0 0 0 0 0 0 1 0 0 0 3 2 1 0 - 3 1 30- 4 2 1 0 1 0 0 0 2 0 0 0 4 0 0 0 F10 0 1 0 0 01- 0 0 0 0 2 1 - 00000 0 00101212309 6 003 1 - 2 0 0 1 F11_0 723 667 1238 1146 1592 2209 571 612 0 0 38 195 525 517 3 1 1 0 1241 761 749 749 1007 1073 277 286 177 190 1083 659 599 667 1091 1123 896 1216 00900 971 385 422 00699 752 01097 001593 9 252 262 431065 2005 682 709 - - 1 6 0 1 F12 - 0 0 0 0 3 3 1 0 2 0 0 1 1 0 - 0 0 0 0 1

0 1 0 00881 143 355 465 01- 6 8 G1_0 6 0 2 0 2 0 2 0 0 0 0 0 5 0 6 0 7 0 0 0 7 0 1 0 0 0 0 0 0 0 2 0 0 0 1 0 3 1 0 0 3 0 100 0 6 0 1 2 0 0 0 0 3013 0 0 0 3 0 4 0 2 0 2 0 308 5 0 0 2 0 0 0 0 1 1 13 1 0 0 3 1 6 0 4 0 9 2 8 1 7 0 0 1 1 2 1 0 0 1 0 1 1 0 0 0 1 0 1 0 4 4 1 1 1 G2 - G3_0 - - - 003 4 0110000 1 - 0 1 220 2 0 2 0 0 2

5 0 0 2 9 05023 4 5 11 030 0 0 0 0 0 0 0 0 0 0 0 5 4 0 0 0 1 2 1 0 0 0 0 1 1 - G4 0 1 0 0 0 0 0 1 0 1 - 10001 5 01- 0 0 0 2 0 0 1 1 5 5 G5 393 344 843 655 2 4 0 3 92 111 1547 1288 339 1321 86 113 2542 2670 920 833 1204 729 989 912 1027 1059 1662 1709 1007 1284 951 476 1023 1249 0 2 0 1 0 2 22 23 0 0 0 0 1191 1327 0 0 0 2 1 2 0 0 0 0 1554 4 11 18 7 12 83 179 164 224 343 538 762 90 1828 377 4 8 0 0 1

0 G6 2243 2794 0 1 1 0 2 1 0 1 - 1 0 0 0 2 1 0 0 0 4 0 0 2 0 6 0 2 1 4 1 2 2 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 - - - - 0 0 259 155 83 357 1 2 1080 1063 1 0 1 1 0 0 0 0 1 4 752 857 716 298 751 934 0 1 6 7 2 0 174 277 311 742 0 0 1 1 829 914 0 0 0 0 3 3 0 0 0 0 1 1 0 0 0 0 0 0 2 0 - 4 0 0 0 2 0 - 0 0 3 13 0 0 1 0 10 5 0 0 1 0 -

- 2 0 1 0 568 499 4 4 680 1333 36 36 68 129 0 1 0 1 0 0 2 5 81 126 - - G9 1254 956 - - 1 0 7 0 0 0 0 0 0 0 0 0 0 0 G8 1 0 2 2 0 1 0 0 4 0 0 0 2 0 6 4 4 0 0 0 1 0 0 0 - 0 0 0 0 0 1 - 3938 4004 - - G7 2948 2786 3 1 1 0 0 0 0 1 1 0 1 0 1 0 4 1 1 1 1 0 1 5 1 0 3 3 G 10 G11 2 0 0 0 4 0 0 0 001 0 1 0 0 0 0 1 0 1 0 0 3 5 4 2 2001100 1

0 0 0 1 0 0 003 0 1 0 0 2649 0 2664 001001000010002164 2932 001010 1004110101765 515 40770000- G12 1339 1322 1112 1947 2 0 1 0 1216 842 0 14 332 457 1842 2367 1 2 355 276 0 0 1031 357 851 889 500 918 0 0 1 0 666 827 38 29 0293 001773 23 2029 2394 0 0 0 0 17 69 1 0 18 15 0 0 433 768 472 367 6 2 1747 2932 5 0 582 684 0 1 2 20 3 0 7 3 1244 1092 0 0 2 1 0 0 0 0 0 0 2 0 1432 1852 0 0 1 0 1439 1847 0 0 1 0 3 0 0 0 2 0 0 0 0 0 H1 - H2 - 2751 3357 1221 1580 2090 2232 - 2147 2713 1 1 1788 1864 547 569

798 502 0 0 0 1 0 2 0 0 - 4010 0 00800 1503 1 0 1 1 404 36 007 2 527 359 1686 1602 - 0 0 5 6 1 0 1419 2683 129 209 0 0 1721 411 1 4 H3 H4 0 2 2 1 1433 1999 0 0 856 1079 184 138 1564 2024 1532 1482 0 0 226 234 0 0 0 3 0 0 1743 790 750 1050 0 2 0 0 0 0 897 1305 0 0 716 845 0 1 0 0 0 0 0 2 0 48 0 0 0 0 1796 2147 0 3 987 2161 809 938 286 584 702 111 189 44 779 1067 795 658 14 9 - - 000 2 0 1 425 499 1723 1668 0 1 2 1 1179 1494 0 3 656 963 151 33 0 2 1012

1325 0 1 1681 2701 134 196 0 1 0 2 0 0 - - - - - 1110 1327 1833 2558 010 0 2363 19 21107 228 1367 1813 1508 2917 1504 293 0 0 2516 4005 39- H5 0000000192 405 010 2 1543 1859 1266 883 556 626 001 0 544 389 79 101 1 0 1256 1815 674 1151 892 1191 10880 1180 441032 1280 908 31423 3002 971 1845 01221308 1154 - H6 0 1 8 1 1 2 0 0 0 1 7 0 - 1 0 0 1 - 0 1 1 1 - - - H7 - 227 3 1 0 00500010222 0 1 4 1 5 00021 0 322 3 0100000 0 2000121200470000- H8 0 0 H9 0 4 02- 0 0 0 0 1260 1832 1323 1823 2 0 - 1555 1120 861 1128 0 1 0 0 0 0 0 0 0 0 1 0 0 2 -

H10 1089 1291 0 0 0 0 1484 2281 0 0 0 0 0 1 0 1 880 1249 0 0 2113 2903 371 833 1287 1807 1 0 575 142 0 4 694 717 - H11 825 869 253 262 0 2 176 253 304 363 189 160 44 152 30 35 1152 1310 681 687 851 502 482 509 230 219 1034 1020 330 380 1 0 695 828 0 0 529 524 0 0 139 183 0 0 9 10 886 1084 0 0 511 513 109 143 0 86 0 0 831 3 71 76 5 8 854 1538 395 461 107 195 717 113 0 0 706 959 0 0 280 341 395 412 285 232 2 0 246 293 266 277 427 351 301 1027 312 357 84 96 207 227 372 258 367 344 0 0 94 76 287 374 936 564 138 124

512 615 1009 1115 742 1169 0 1 157 347 H12 1 1 1 0 0 0 0 2 5 252 2 3 3 6 0 1 1175 1780 0 0 0 0 1 2 0 0 313 1117 0 0 385 715 0 0 450 564 983 1064 77 68 243 309 0 0 0 1 409 3 974 1190 0 4 0 0 191 302 0 0 187 1211 338 603 647 749 55 91 736 239 1143 298 104 148 730 601 331 319 137 630 791 786 497 785 0 1 Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Which Epitopes go into the vaccine? 1) Diversify across HLA alleles 2) Select mutations with balanced allele specific expression 3) Maximize (expected) clone coverage 4) Include epitopes with high MHC binding affinity 5) Include epitopes with high Differential Agretopic Index (DAI): difference in MHC affinity between mutant epitope and its wild type counter part Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Duan et. al., JEM 2014 Sequencing QC and

Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Expitope (Haase et. al., Bioinformatics 2014) Checks for cross-reactivity based on ENCODE RNA-Seq from normal tissues http://webclu.bio.wzw.tum.de/expitope/ OptiTope (Toussaint and Kohlbacher, Nucleic Acid Research 2009) Optimizes allele coverage http://etk.informatik.uni-tuebingen.de/optitope Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing Toussaint and Kohlbacher, Nucleic Acid Research 2009 Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing T Cell Receptor sequencing Compare the TCR repertoire before and after immunization to determine response against used epitope(s) Primary analysis of TCR sequencing data IMSEQ (Kuchenbecker et. al., Bioinformatics 2015) http://www.imtools.org/ HTJoinSolver (Russ et. al., BMC Bioinformatics 2015) https://dcb.cit.nih.gov/HTJoinSolver/ Sequencing QC and Mapping Calling SNVs Epitope Prediction Clonality Analysis Vaccine Design TCR Sequencing tcR (Nazarov et. al., BMC Bioinformatics, 2015) R package for downstream analysis, including diversity measures, shared T cell receptor sequences identification http://imminfo.github.io/tcr/ THANK YOU! QC FASTX: http://hannonlab.cshl.edu/fastx_toolkit/) PRINSEQ: http://prinseq.sourceforge.net/) Epitope prediction NetChop: http://www.cbs.dtu.dk/services/NetChop/ SYFPEITHI: http://www.syfpeithi.de/ NETMHC: http://www.cbs.dtu.dk/services/NetMHC/ NetCTL: http://www.cbs.dtu.dk/services/NetCTL/ Tumor Specific epitope predicton pipeline Epi-Seq: http://dna.engr.uconn.edu/?page_id=470 Also available on out galaxy server: http://mhc1.engr.uconn.edu:8080/ Vaccine design Expitope: http://webclu.bio.wzw.tum.de/expitope/ OptiTope: http://etk.informatik.uni-tuebingen.de/optitope TCR sequencing analysis Epi-Seq: http://imminfo.github.io/tcr/

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