1. Computational methods for genomics-guided immunotherapy Sahar Al Seesi Computer Science & Engineering Department, UCONN Immunology Department, UCONN Health

2. Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing

3. Ion Proton Tumor DNA Exome AmpliSeq Normal DNA Tumor RNA Sequencing Whole Transcriptome Library prep Whole Genome Library prep or Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing

4. Tumor DNA Sequencing Nextera Rapid Capture Exome Normal DNA Tumor RNA Whole Transcriptome Library prep Illumina HiSeq Whole Genome Library prep or Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing

5. Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing CCGCGGTCAG TCTCGGAATT TCCCGGTAAT TCCCGGTAAT TCCCGGTAAT ATCGGTTTAT CCTCTAACAC TAATGGAATT CCGCGAACAC AACACCCCGG Read Quality Control Low quality base

6. Tools to analyze and preprocess fastq files – FASTX http://hannonlab.cshl.edu/fastx_toolkit/ http://hannonlab.cshl.edu/fastx_toolkit/ – PRINSEQ http:// prinseq.sourceforge.net /) http:// prinseq.sourceforge.net / Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing

7. Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing Normal Exome Reads Tumor Exome Reads Tumor RNA-Seq Reads Human reference Read Mapping

9. Normal Exome Reads Human Reference Tumor Exome Reads Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing * * * * * * * * *

10. Somatic Variant Callers Mutect (Broad Inst.) VarScan2 (Wash. U.) SomaticSniper (Wash. U) Strelka (Illumina) SNVQ w/ subtraction (UConn) Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing Normal Exome Reads Human Reference Tumor Exome Reads * * * * * * * * *

11. 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 IonTorrent Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing

12. Criteria for selecting candidate epitopes 1)Gene harboring the SNV must be expressed (FPKM estimation) Sequencing QC and Mapping Calling SNVs Epitope Prediction TCR Sequencing Tumor RNA-Seq Reads ** * *

13. 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 TCR Sequencing