Chromium™ Single Cell V(D)J Solution

Revolutionizing Applied Immunology With Immune Repertoire Profiling at Single Cell Resolution

May, 2017

© 10x Genomics, Inc. 2016© 10x Genomics, Inc. 2017 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Confidential — Do not distribute The Opportunity for V(D)J

•The V(D)J Solution will Revolutionize Biology: –Basic and Translational Research – Human Health and Disease –Immunotherapy –Vaccine Development

•The V(D)J Solution supports critical immune repertoire profiling applications: –Assess the diversity / clonality of the immune repertoire –Track specific T-cell clones over time and/or between samples –Identify immune receptors (antigen specificity) shared between people

•Full-length paired single-cell sequencing of TCR α / β transcripts is critical for: –understanding the true biological diversity of T-cell (antigen) receptors –elucidating the molecular genetic determinants of antigen specificity

2 T-Cells Carry Out Cellular Immune Responses

T cells attacking a tumor cell http://www.diamond.ac.uk/Home/News/LatestFeatures/02_09_15.html 3 T-Cells are Selected for Specificity of Response

• Positive selection for MHC (HLA type) • Negative selection for failing MHC recognition • Negative selection for specificity against self

• This process results in a T-cell repertoire selected for specificity for “non-self” antigens presented on MHC.

https://www.slideshare.net/jugafoce/immune-15100391

4 T-Cell Selection and Function

• T-cell selection requires two features: • Heterodimeric cell-surface T-cell receptors (TCRs: TCRa and TCRb) • antigens presented by MHC

• In healthy individuals, TCRs are highly diverse • Estimated >1015 possible combinations • “True” diversity is a current research question

5 T-Cell Diversity is Created By V(D)J Recombination

• TCRb has Variable (V), Diversity (D) and Joining (J) gene segments • TCRa has Variable (V) and Joining (J) gene segments

• V(D)J refers to combined TCRa/b recombination

• TCRb has ~50 V x 2 D x 12 J segments = ~1,200 possible combinations (103)

• So…where does 1015 diversity come from?

6 T-Cell Diversity is Created By V(D)J Recombination

• Untemplated diversity (indels) not encoded by the germline is generated at recombination junctions (N) between Vaja and VbDb / DbJb

• This untemplated nucleotide diversity creates the massive potential combinatorial diversity of the adaptive immune repertoire

• T-cell receptor diversity is encoded by Complementarity-Determining Region 3 (CDR3)

7 V(D)J Recombination Creates Clonal “Barcodes”

8 The Adaptive Immune System

• V(D)J Recombination creates both T-cell and B- cell receptor (antibody) diversity

9 Methods for Immune Repertoire Profiling – Single Chain

•Spectratyping •RT-PCR and NGS •Multiplex PCR and NGS –Multiplex single-chain –Uses universal sequences for –Flexible sample types, no (TCRb) PCR amplification enrichment required –Bands-on-gel visualization –Typically measures expression –Can enable quantitative –Low cost and low resolution rather than true clonality measurements –Very poor gauge of diversity –Single-chain (e.g. TCRb) –Single-chain (e.g. TCRb)

Characterizing immune repertoires by high throughput sequencing: strategies and applications http://dx.doi.org/10.1016/j.it.2014.09.004 10 Methods for Immune Repertoire Profiling – Paired Chains

• Advanced Methodologies in High-Throughput Sequencing of Immune Repertoires Simon Friedensohn et al Trends March 2107 11 The Chromium™ Single Cell V(D)J Solution

•Comprehensive, scalable solution for profiling full-length, paired V(D)J transcripts from 100 to 100,000 Human T-cells –Human B-cells to be supported later in 2017

•Built from proven components –Chromium Single Cell Controller –Robust consumables and reagents adapted from the Chromium Single Cell 3’ Solution –Cell Ranger software with new V(D)J analysis module –Loupe VDJ visualization software

12 Single T-Cell Partitioning, Lysis and Barcoding

V(D)J Proﬁling of Individual T-cells

Cell 1...

TCRα TCRβ

Cell 5,000 TCRα TCRβ

• Output: Digital V(D)J profiles from • Rapid partitioning and lysis of T-cells partitioned T-cells in < 7 minutes • Lowest cell loss

13 Chromium Single Cell 3’ Assay Scheme

•UMI and Cell Barcode adjacent to poly(dT)VN RT primer

•TSO at 5’ end of RT product

•TSO and RT primer contain sequences for universal PCR amplification

14 Chromium Single Cell V(D)J Assay Scheme

• RT enzyme and poly(dT) primer delivered to all GEMs as part of master mix • Barcoded 5’ template switch oligo delivered to GEMs from Gel Beads • RT reaction generates unbiased cDNA with a sequencing adapter, a cell barcode and a UMI on the 5’ end

• Enrichment PCR with a universal primer for the 5’ adapter and successive nested primers for the TCR constant regions.

• Fragmentation and sequencing optimized for assembly of the full V(D)J sequence (5’ UTR to constant regions) from short paired-end reads on a cell-by-cell basis.

15 Advantages of Chromium VDJ Solution • Less Bias –No V- or J-specific primers, avoids bias associated with complex multiplex PCR –UMI in template switch oligo further reduces sequencing error and enables transcript quantitation

• More Discovery –Detect germline and somatic variants across the entire V(D)J segment –See both productive and expressed non-productive recombination events –See multiple TCR alpha genes expressed per cell –Map transcription start sites

• Flexible Reagents –One gel bead for any species, cell type and isotype –Enables future capability to assay additional transcripts for cell type classification or phenotyping

16 New Single Cell A Chip Kit - Rapid and Efficient Microfluidics

•Partition 100-10,000+ cells per channel in < 7 minutes •Run 1 to 8 channels in parallel •No lower size limit on cells •Recovers up to 65% of all loaded cells, including: –T cells, B cells, PBMCs and cell lines –FACS-isolated cells –MACS MicroBead-enriched cells –Tumor-infiltrating lymphocytes •Low doublet rate: 0.9% per 1,000 cells

17 Compatibility with all Illumina Sequencers

•Choose the optimal sequencer for the scale of your experiments*

•2x150bp sequence reads, 8bp i7 Index

•5,000 read pairs/cell for V(D)J assembly**

*Single Cell V(D)J libraries on the NextSeq 500/550 platform may yield reduced sequence quality and sensitivity relative to the MiSeq and HiSeq platforms. ** Recommended starting point. Optimal depth may vary across cell types and applications. 18 Cell Ranger™ - Informatics Workflow

• Complete Linux-based software package • Runs anywhere: Local Mode and Cluster Mode • Widely used for single cell gene expression analysis • Version 2.0+ includes V(D)J analysis and support for Loupe VDJ visualization module

Transcript counting pipeline

BAM, Cell-by-Cell Clonotype Barcode Read filtering Annotation FASTQ, Consensus Inference and Processing [V(D)J] [V(D)J] HTML, Assembly Clustering CSV Output File Formats

19 Chromium™ Single Cell V(D)J Solution workflow

• Sequence Chromium libraries to ~5,000 read pairs per cell (2x150 nt) • Cell Ranger™ pipeline assembles reads into V(D)J segments at single cell resolution • Loupe™ V(D)J Browser enables interactive analysis

BAM FASTQ Standard Informatics Python, R CSV BCL Cell Ranger™ Analysis Pipelines

Loupe™ VLOUPE V(D)J Browser Interactive Analysis 20 Pricing

Product PN Units List Price

Chromium™ Controller, 24 Mo. Warranty 120246 1 Instrument $125,000

Chromium™ Single Cell Controller, 24 Mo. Warranty 120212 1 Instrument $75,000

Chromium™ Single Cell A Chip Kit 120236 48 samples $1,440

Chromium™ Single Cell 5’ Gel Bead & Library Kit 1000006 16 reactions $20,000*

Chromium™ Single Cell V(D)J Enrichment Kit, Human T Cell 1000005 96 reactions $250

Chromium™ i7 Multiplex Kit 120262 96 reactions $768

130002 Platform Assurance Plan (Controller / Single Cell Controller) 12 months $12,500 130004

*Price per cell $0.13 to $1.20 21 The Chromium™ Single Cell V(D)J Solution • Paired, full-length V(D)J immune repertoire analysis for every lab • Built on proven microfluidics and software • Turn-key solution that doesn’t require extensive optimization • Flexible Platform • Support for Human B-cells and other species coming soon • High capture efficiency for precious samples • Up to 65% of loaded cells can be recovered from samples containing only 100s of cells

• High throughput for deep repertoire profiling • Capture 80,000+ cells in <7 minutes

22 The Chromium Single Cell V(D)J Solution

Data

Confidential — Do not distribute 23 Human T cells selected for Tetanus Toxoid Specificity in vitro

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 469 81% 96% 78% 1.2

Only one productive α and one productive β chain observed in the library: α chain consensus (589nt) TRAV12-2:J43 AGTTTTCTTATATGGGGAAAGCAGATTCTTTTTATGATTTTTAAAGTAGAAATATCCATTCCAGGTGCATTTTTTAAGGGTTTAAAATTTGAATCCTCAGTGA ACCAGGGCAGAGAAGAATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAAT TCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGA AAAGCCCTGAGTTGATAATGTCCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCAT CAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACATCAATAACAATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAACCA AATATCCAGAAGCCTGACCCTGCCGTGTACCAGCTGAGAGACTAGATCGGAAGAGCAGATGTACCCAGGATATG β chain consensus (610nt) TRBV6-4:D1:J2-3 TTTCTCATCTGGGGCTCTCTCGACAAGAAGGTTCTGGGGACCAGGCAGAGAGAATGAGGTCTCAGGATGACTTCCTTGACAGCCCTGTTCCCCTTTCATCAAC ACACAGACCCAGAAGACCTCTCTGTCTTGTAGCATCTGCCATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGATTGCT GGGATCACCCAGGCACCAACATCTCAGATCCTGGCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACATAATGCCATGTACTGGTATAGAC AAGATCTAGGACTGGGGCTAAGGCTCATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAGTCCCTGATGGTTATAGTGTCTCCAGAGCAAACAC AGATGATTTCCCCCTCACGTTGGCGTCTGCTGTACCCTCTCAGACATCTGTGTACTTCTGTGCCAGCAGTGACTCGGGGCTTCTCACAGATACGCAGTATTTT GGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAGATCGGAAGAGCACA • Consensus accuracy across V(D)J region: 100% • 98.8% of chains from individual cells of this clonotype are identical to this V(D)J consensus • Sequence accuracy from individual cells of this clonotype: 99.98% (1.2 errors in 10,000 nt) 24 Human T cells selected for Epstein-Barr Virus Specificity in vitro

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 446 83% 66% 53% 4.6

Top 10 detected TCRs: count

• ~55% of cells inferred to share one major α and β chain (TRAV12-3:J20, TRBV9:D1:J1-4)

25 Human T cells selected for Epstein-Barr Virus Specificity in vitro

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 446 83% 66% 53% 4.6

Top 10 detected TCRs: count

• ~9% of cells inferred to be of a second, unrelated clonotype (TRAV5:J15, TRBV14:D2:J2-1)

26 Human T cells selected for Epstein-Barr Virus Specificity in vitro

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 446 83% 66% 53% 4.6

Top 10 detected TCRs: count

• ~4% of cells of two related clonotypes that share a common β chain: ~3% of cells: TRAV5:J15 (CDR3: CAESSNQAGTALIF) + TRBV29-1:D1:J1-4 (CDR3: CSVGQGGTNEKLFF) ~1% of cells: TRAV5:J23 (CDR3: CAESIGKLIF) + TRBV29-1:D1:J1-4 (CDR3: CSVGQGGTNEKLFF) 27 Peripheral Blood Mononuclear Cells from the Same Donor (x2)

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 3,424 55% 85% 50% 1,391.9 Sample #1 3,621 62% 84% 57% 1,437.6 Sample #2

No clonotype makes up >0.5% of either sample

Clonotypes unique to Sample #1 Clonotypes unique to Sample #2

2,809 91 2,949

Clonotypes detected in both

• Tetanus toxoid- and EBV-specific clonotypes not observed

28 Representation of Germline V/J Segments

Germline V segments Germline J segments contig Median UMI count/

TCR α TCR β TCR α TCR β

29 Limit of Detection: EBV-specific T cells spiked into PBMCs

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 3,794 64% 86% 59% 1,223.2 Mix #1 (1% EBV: 99% PBMC) 2,067 50% 83% 45% 933.0 Mix #2 (1% EBV: 99% PBMC)

~55% of EBV-specific cells: TRAV12-3:J20 (CDR3: CATQGSNDYKLSF), TRBV9:D1:J1-4 (CDR3: CASSTGQVATNEKLFF) → ~0.55% of cells in 1:99 PBMC mixes should have this major clonotype

• 16 cells (0.4%) with identical α or β detected in Mix #1 (11 paired) count

• 7 cells (0.3%) with identical α or β detected in Mix #2 (3 paired) count

30 Limit of Detection: EBV-specific T cells spiked into PBMCs

~9% of EBV-specific cells: TRAV5:J15 (CDR3: CAESSNQAGTALIF), TRBV14:D2:J2-1 (CDR3: CASSQSPGGIQFF) → ~0.09% of cells in 1:99 PBMC mixes should have this second clonotype

• 2 cells (0.05%) with identical α or β detected in Mix #1 (2 paired) count

• 1 cells (0.05%) with identical β detected in Mix #2 (0 paired) count

→ Limit of Detection for a known clonotype among 2,000+ cells is <1% 31 Profiling T cell sub-types from healthy peripheral blood

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 4,827 71% 91% 65% 2,850.8 CD3+ T Cells 4,908 75% 94% 70% 3,290.4 CD4+ Helper T Cells 4,810 64% 91% 58% 1,178.6 CD8+ Cytotoxic T Cells

Top 10 detected TCRs: count

• No clonotype makes up >0.1% of the sample 32 Profiling T cell sub-types from healthy peripheral blood

Top 10 detected TCRs: count

• Evidence of individual T cells expressing two productive α chains 33 Profiling T cell sub-types from healthy peripheral blood

Top 10 detected TCRs: count

• Top clonotype makes up ~1% of the sample 34 Tumor Infiltrating Lymphocytes (TILs)

Cryopreserved dissociated tumor CD3+ TILs

Colorectal cancer (CRC)

• Primary tumor Dead cell depletion • Stage II-A, LV-0, R-0 Anti-CD3 bead enrichment • Female, white, 67 y.o.

Clear cell renal cell carcinoma (CCRCC) • Primary tumor • Stage 1 Dead cell depletion Anti-CD3 bead enrichment • Male, white, 54 y.o.

35 Tumor Infiltrating Lymphocytes (Anti-CD3 Bead Enrichment)

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 2,019 47% 90% 44% 445.3 CRC 755 73% 90% 67% 15.0 CCRCC

Top 10 detected TCRs: count

• ~0.8% of cells make up most abundant clonotype (TRAV20:J15, TRBV29-1:*:J2-5) • Clonotype diversity is similar to circulating T cells

36 Tumor Infiltrating Lymphocytes (Anti-CD3 Bead Enrichment)

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 2,019 47% 90% 44% 445.3 CRC 755 73% 90% 67% 15.0 CCRCC

Top 10 detected TCRs: count

• ~21% of cells inferred to share one major α and β chain (TRAV13-1:J5, TRBV27:D1:J1-1) • Suggests significant clonal expansion of TILs in this tumor

37 Tumor Infiltrating Lymphocytes (Without Enrichment)

Cells with TCR(s) Productive α Productive β Productive pair Clonotype detected detected detected detected diversity (Inverse Simpson index) 755 73% 90% 67% 15.0 CCRCC (Anti-CD3 bead enriched) 190 62% 72% 58% 8.0 CCRCC (No enrichment)

• Enriched TILs top clonotype: ~21% frequency (TRAV13-1:J5, TRBV27:D1:J1-1) • Unenriched TILs top clonotype: ~20% frequency (TRAV13-1:J5, TRBV27:D1:J1-1)

→ Full-length, paired V(D)J segments from clonally expanded TILs without cloning or ex vivo culture α chain consensus (682nt) TRAV13-1:J5 TGTCTTATATGGGGATTGGTTGGCTACACAGTGTGAGAAACCCCTATGGCTGCCAGAGGAGAGAAGAGACAACCTGATGATAGAAGTAACTCTTATAACTGGA GGTTGCAGGTCAATGACTGATCTTAATTGGGAAGAACAAGGATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGAATGGAGA GAATGTGGAGCAGCATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAACTACTTCCCTTGGTAT AAGCAAGAACTTGGAAAAAGACCTCAGCTTATTATAGACATTCGTTCAAATGTGGGCGAAAAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCA AACATTTCTCCCTGCACATCACAGAGACCCAACCTGAAGACTCGGCTGTCTACTTCTGTGCAGCACCCCGCCGGGGCAGGAGAGCACTTACTTTTGGGAGTGG AACAAGACTCCAAGTGCAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCCCGGATGAAGCTACAAGCGCGAGTGACGTTATCTGAT TTGATGGAAGAAATCTGAAGCTGGATGAGGGCCAAGCGGAAAGAGGTCATGACTCTGCAGACAA β chain consensus (563nt) TRBV27:D1:J1-1 TATCTTATATGGGGGTTTCTGAGGCCCAAATAGCTGAAGAGGTGGAGACGTTACAGAAACCACCTGGAGCCCCCAGAACTGGCAGACACCTGCCTGATGCTGC CATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACT GGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAA TGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAA CCAGACCTCTCTGTACTTCTGTGCCAGCAGTTTAGGACAGAACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGGTG TTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAGATCGGAAGAGCA 38