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UCSC Immunobrowser Hyunsung John Kim 10/16/2013. UCSC Immunobrowser Analyze status of Adaptive Immune System Tracks T-cells based on sequence signature.

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Presentation on theme: "UCSC Immunobrowser Hyunsung John Kim 10/16/2013. UCSC Immunobrowser Analyze status of Adaptive Immune System Tracks T-cells based on sequence signature."— Presentation transcript:

1 UCSC Immunobrowser Hyunsung John Kim 10/16/2013

2 UCSC Immunobrowser Analyze status of Adaptive Immune System Tracks T-cells based on sequence signature in the T-Cell Receptor Beta Targeted Next Generation Sequencing generates billions of data points Can we provide Repertoire-level analysis while preserving Receptor-level detail Search the literature for clues on T-cell function

3 Adaptive Immune System Specific – Receptors to bind exclusively to a pathogen Diverse – Receptors can be designed to almost any pathogen Memory – Predesigned receptors are reused for subsequent infections Self/non-self recognition – T-Cells/B-Cells do not attack healthy self cells

4 Specific and Diverse Receptors Each receptor has a unique genome sequence

5 Memory

6 What do B-cells and T-cells do? http://www.unm.edu/~mpachman/Blood/t_cel l_killing_cancer_cell.jpg http://www.hu.liu.se/ike/forskning/cellbiologi/ros en-anders/rosen/1.264183/Bcell200.jpg B-cellHelper T cell (T H )Cytotoxic T-cell (T C ) Mature in Bone Marrow Binds directly to antigen Secretes soluble antibodies Antibodies aggregate antigens, mark them for phagocytosis or lysis via the complement system Mature in Thymus CD4+ Binds to peptide-MHC class II molecules present only on Antigen Presenting Cells (APCs) Activates B-cells Regulates Immune response Mature in Thymus CD8+ Binds to peptide-MHC class I molecules present on most nucleated cells Destroys infected cells Sciencephoto.com 6

7 Antibodies and the TCR belong to the Antibody-fold superfamily Goldsby et al, 2007 7

8 Antibodies and the TCR belong to the Antibody-fold superfamily Goldsby et al, 2007 8

9 Complementarity Determining Regions (CDR) determine binding specificity 9 Tertiary: Secondary: Primary:

10 Specific and Diverse Receptors CDRs

11 Random Receptor Sequence generated through VDJ Recombination ~10 15 -10 20 Distinct TCR sequences Diversity generated through: – Combinatorial Joining (left) – Random Nucleotide addition

12 Combinatorial Joining of VDJ Gene Segments LocusVDJTotal IgH38-462366348 IgK34-3805190 IgL29-3304-5165 TRA43-450502300 TRB40-48212-132300 12 BCR Recombination Total = (IgH x IgK) + (IgH x IgL) = (6348 x 190) + (6348 x 165) = 2.25 x 10 6 TCR Recombination Total = TRA * TRB = 2300 x 2300 = 5.29 x 10 6

13 Insertions and Deletions further diversify TCRB and IGH

14 Allelic Exclusion and Selection ensures self/non-self recognition and functional T- cells Jung et al, 2006 14

15 Clonal Expansion Shapes the Repertoire 15 http://163.16.28.248/bio/activelearner/43/ch43c3.html

16 What is an Immune Repertoire? Set of all BCR and TCR sequences in an individual – 10 11 B-cells and T-cells 16

17 Immunosequencing Workflow Data Analysis Sequence Recombined Regions with HTS Amplify Recombined Regions Enrich cells for B/T lymphocytes Blood Draw 17

18 Cell-level Enrichment Sorting techniques – Fluorescence activated cell sorters (FACS) – Magnetic activated cell sorting (MACS) CD Receptors distinguish lymphocytes MarkerTarget CD3 CD4 CD8 CD19 CD20 CD45RA CD45RO T-cells Helper T-cells Cytotoxic T-cells B-Cells Naïve T-cells Memory T-cells 18

19 Input Material DNA DNA is stable DNA-primers can also be used on cDNA Non-productive Rearrangements Long Introns require specific primers for V and J gene segments RNA RNA is unstable Requires cDNA synthesis Allelic exclusion enriches for expressed receptors Spliced transcripts allow for constant region primer(s) 19

20 Assay Modified from Warran, et al 2009 20

21 High throughput sequencers 454 Titanium Long reads – ~600bp reads Medium throughput – 10 6 reads Preferred for B-cells Illumina HiSeq Short reads – 2x100bp Paired-end reads High throughput – 3 x 10 9 reads Preferred for T-cells 21

22 Junctional Analysis

23 VJ gene segment usage Wang et al, 2010 Boyd et al, 2009 Robins et al, 2009 Weinstein et al, 2009 23

24 UCSC Immunobrowser Takes in annotated sequence data Generates Repertoire-level visuals Persistent filters view subsets of a repertoire Track clonal expansions over multiple samples – Identify aberrant clonal expansions (Blood cancers) – Track clonal expansions (Immune response) Search literature RESTful urls makes sharing easy (Dev) http://laozi.soe.ucsc.edu:8000http://laozi.soe.ucsc.edu:8000

25 The Future Motif Finding Group vs group comparisons The personal sequencer – Regular TCR/IG sequencing Integration with Medbook-like service – Detect reoccurence of disease or relapse of autoimmune disease – Know if you’ve got the flu or a cold

26 Fin. Thanks to: – Max Haeussler – Ngan Nguyen – You, for your time Questions?


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