Introducing the Chromium™ System

One system, one workflow, powerful new sequencing applications10xGenomics.com

The Chromium™ System

One system, one workflow, powerful new sequencing applicationsGemCode™ technology powers an innovative system that transforms the capability of existing short-read sequencers. With millions of uniquely addressable partitions, the Chromium System unlocks critical genomic information.

Genome SequencingThe Chromium Genome provides long range information on a genome-wide scale, including variant calling, phasing and extensive characterization of genomic structure. Unlock critical genetic information for variants in heritable disorders, and discover key alterations in cancer.

Single Cell Sequencing With Chromium Single Cell 3’, perform deep profiling of complex cell populations with high-throughput digital gene expression on a cell-by-cell basis. Trace expression profiles to individual cells to ensure biologically relevant signals are not masked by bulk average measurements.

Targeted SequencingThe Chromium Exome provides long range information, enabling phasing, structural variant detection and copy number determination. Low complexity and repetitive regions previously missed with short-read sequencing are now accessible.

De Novo AssemblyDiscover the true genome with the Supernova™ Assembler and open the door to low-cost, everyday diploid assemblies. Unlock sample- specific sequence, probe diploid genome structure, and remove the need for a reference sequence of any kind.

The Chromium™ SystemUnlock critical long range genomic and cell-by-cell gene expression information.

Changing the Definition of Sequencing™10x Genomics

1 2

Chromium™ Genome

The most comprehensive genomeLong range genomes at population scale. Call and phase the full spectrum of variants and unlock previously inaccessible regions from a single library.

Massive partitioning and complete genomic information from a single library with 1ng inputThe Chromium Genome performs massive partitioning and barcoding of 1ng input DNA. The system produces uniform, high-complexity libraries, compatible with Illumina’s HiSeq X Ten systems, enabling high quality variant calling from minimal sequencing depth.

Resolve the genome into multi-megabase phase blocksWhile human genomes are diploid, analysis based on short reads collapses variants into a single haploid call set, masking critical genetic relationships. Chromium Genome phases the full spectrum of variants (SNVs, indels, and large-scale structural rearrangements) into ultra long multi-megabase phase blocks, enabling a full understanding of diploid genome sequence.

Uncover previously inaccessible parts of the genome and hidden variants in critical genesShort reads cannot be mapped into repetitive regions, causing regions with no coverage (Child: BWA Aligner). The Lariat™ Aligner uses Linked-Read information to map reads that could not normally be mapped (Child Lariat Aligner), enabling variant calling and phasing in regions normally inaccessible to short reads. Father: NA12891 Child: NA12878 Mother: NA12892.

Chr 6

Hap 1

Hap 2

50 Mb Phase Block

70 kb Deletion

Partitions

Barcode Pool

Input DNA

384

STANDARDBARCODING

384

100ng+

>1,000,000

Chromium™

Genome

4,000,000

1ng

Detection of a SNP in RPS17. The Lariat Aligner uses barcode information to correctly map reads and call variants in a highly repetitive region that standard aligners miss due to low mapping quality.

A region in chromosome 3 is represented in the diploid assembly of NA12878 by two >10Mb phased scaffolds within a larger 17Mb overall scaffold segment. A zoom into the figure shows that variants between the haplotypes are conserved in the assembly such as the 8kb deletion indicated, where traditional assemblies compress the two haplotypes into a single path.

Multi-megabase diploid assemblies eliminate the need for a referenceThe Supernova™ Assembler utilizes Linked-Reads to de novo construct multi-megabase diploid assemblies, preserving phasing information for small variants, structural rearrangements and novel sequence.

ChildBWA Aligner

Child

Mother

Father

RPS17 Exon 3Chr15

MotherLariat Aligner

FatherLariat Aligner

ChildLariat Aligner

G ACG

A G

CG

A G

G A GA G G

A G

MAPQ>30

Changing the Definition of Sequencing™10x Genomics

3 4

Chromium™ Exome

Reach beyond the exomeThe Chromium Exome with optimized baits powered by Agilent’s market-leading SureSelect® technology enables access to low complexity and duplicated regions, megabase-scale phasing, structural and copy number variation while improving the quality of SNP calls.

Enable phasing of thousands of genes and detection of structural and copy number variationThe optimized SureSelect baits are designed specifically for the Chromium Exome to improve gene phasing by closing gaps, and recovering hard-to-map loci in the genome.

Map previously inaccessible regions and improve variant calling performanceThe barcode-aware Lariat™ Aligner maps reads in previously inaccessible regions, identifies new variants and eliminates false positives due to incorrect mapping.

Identify structural variantsDetect gene fusions, duplications, deletions and translocations from a single exome run.

7,693 bp

NA12878BWA Aligner

NA12878Lariat Aligner

RPS17

Optimized SureSelect Baits

MAPQ>30

TRPV1 SHPK CTNS P2RX5-TAX1BP3

57kb Deletion

Hap1

Coverage

Hap2

G>A

20 291 6

EML4-ALK

EML4 ALK13 Mb

BCOverlap

Coverage

Linked Reads

In collaboration with

Exome Baits

Linked-Reads from Exome Baits

Optimized SureSelect Baits

Linked-Reads from Chromium Exome

Resolve compound heterozygosity and copy number changeEstablish cis or trans relationships between variants without trio sequencing.

Changing the Definition of Sequencing™10x Genomics

5 6

Chromium™ Single Cell 3’

High-throughput single cell RNA sequencingSingle cell resolution is the key to uncovering the true complexity of heterogeneous populations. Chromium Single Cell 3’ provides scalable transcriptional profiling of 1,000s to 10,000s of individual cells.

Profiling 1,000s to 10,000s of cells per experiment increases sensitivity and accuracy for the detection of rare cell types.

t-SNE projection of gene expression profiles from 68,000 unsorted human peripheral blood mononuclear cells (PBMCs). Colors indicate the closest match in a reference panel of sorted cell types.

Multiple key applicationsImmunologyNeurobiologyCancer BiologyStem Cell ResearchEmbryology

High-throughputUp to 8 channels processed in parallel1,000 to 6,000+ cells per channel10 minute run time per chipNo cell size restrictions~50% cell processing efficiency

60,000

60,000

Human transcript counts

Human:MouseHuman onlyMouse only

Mou

se tr

ansc

ript

cou

nts

0

Number of distinct genes detected per cell as a function of sequencing coverage for HEK293T cells. Graph shows mean (black line) and range (dark gray) over 16 independent experiments.

Approximately 1,400 human (HEK293T) and mouse (NIH3T3) cells were mixed at a 1:1 ratio prior to profiling. 99.4% of the resulting cell-containing GEMs gave rise to sequencing reads mapping to only one species. This implies a total doublet rate of 1.0%.

Proliferating HEK293T cells were profiled and scored for expression of markers associated with each major cell cycle phase. Cells from all phases were identified at expected proportions.

Efficient biochemistry captures thousands of genes per cell

Single cell partitioning with low multiplet rates

Differential expression identifies cell cycle phase

Changing the Definition of Sequencing™10x Genomics

7 8

GemCode™ Technology Process

1Creating the GEMs

Gel beadFunctionalized with defined DNA barcodes.

Mega diversity reagent libraries.

Up to 100 million barcoded oligos per bead.

Deformable polymeric beads dissolve on demand.

DropletMonodisperse water-in-oil droplets.

Massively scalable to millions of partitions.

Together make a GEMGelbead in Emulsion.

Uniquely addressable reactions for molecular barcoding on a massive scale.

>90% of all droplet reactions receive a single barcoded gel bead.

2Implementing the GEMs

Disposable microfluidicsHighly automated reaction assembly.

Millions of “effective” pipette steps per minute.

On a single instrumentTouch screen operation. ~10 minute run time.

3Discovering with GEMs

Definitive resultsSingle molecule resolution. Single cell analysis on a massive scale.

Make the Discovery with GemCode™ Technology

GemCode™ Technology with millions of uniquely addressable partitions enables single molecule resolution and single cell sequencing

Gel bead in emulsion (GEM)Barcode and reagent delivery with sample partitioning on a massive scale.

Changing the Definition of Sequencing™

10

10x Genomics

9

Barcoded Fragment

Embedded Barcode

Barcoded cDNA

Embedded Barcode

Cell Cell

Cell Cell

Gene 1 Gene 2... Gene 2,000

Gene 1 Gene 2... Gene 2,000

Cell 1...

Cell 5,000

Partitioning & Molecular Barcoding at Scale

Partition sample across 100,000s to 1,000,000s of partitions, each containing a unique barcode

DNA Linked-Reads

Single Cell Digital Gene Expression

Barcoded Primer Library

GEMs Collect Pool

Enzyme

DNA or Cells Oil

Genome is replicated and barcoded via a low-level enzymatic replication.

mRNA is transcribed by a reverse transcriptase that creates barcoded cDNA.

A barcode identifies transcripts originating from a single cell, which are then counted.

Gel beads loaded with barcoded oligonucleotides are first mixed with sample and enzyme mixture, then combined with an oil-surfactant solution at a microfluidic ‘double-cross’ junction.

Gel bead-containing droplets (GEMs) flow to a reservoir and are harvested. Gel beads are dissolved and molecular barcoding of the sample is initiated. The barcoded products are pooled from each droplet. Standard library construction adds the remaining sequencing adapters and sample indices.

Lines represent Linked-Reads. Dots represent reads. Color indicates barcode. Reads with same barcode originated from molecules encapsulated in the same partition.

Changing the Definition of Sequencing™

12

10x Genomics

11

Building upon widely accepted aligners and variant callers such as BWA, GATK, and Freebayes, the Long Ranger pipelines leverage the Chromium System’s molecular barcoding to enable phasing and structural variant calling.

Starting with pre-built or user-supplied transcriptomes, the Cell Ranger pipelines can process cellular barcodes to produce gene expression profiles across tens of thousands of cells at once.

The Supernova Assembler exploits Linked-Read data to reconstruct a diploid genome, unlocking sample-specific sequence and removing the need for a reference sequence of any kind.

The Martian language is complemented by a type-checking compiler, visual IDE, and debugging and profiling tools. The Martian runtime provides built-in support for pipeline composability, parallelization, and parameter sweeping.

Long Range Genomes and ExomesLong Ranger™ Analysis Pipelines

Leverage molecular barcoding to generate a powerful data type called Linked-Reads. Novel algorithms in the Long Ranger pipelines yield several classes of biological insights from Linked-Reads:

• Barcode-aware alignment• Megabase-scale haplotype phasing• Structural variant detection and phasing• Greater accuracy in SNP and indel calling

Single Cell TranscriptomicsCell Ranger™ Analysis Pipelines

Turn-key analysis for single cell gene expression sequencing data. Combining robust transcriptome alignment with large scale cellular barcoding, the Cell Ranger pipelines rapidly generate expression profiles to shed light on:

• Complex primary cell populations• Cellular heterogeneity in cancer tumors • Cell cycle behavior

De Novo AssemblySupernova™ Assembler

The Chromium System opens the door to low-cost, everyday diploid genome assemblies. The Supernova Assembler leverages the unique properties of Linked-Read data to reconstruct the genome under study, without the need for a reference genome.

Open Source InformaticsMartian™ Technology

Chromium Software is built using a powerful, open source informatics platform called Martian. The Martian language features an elegant syntax for defining complex pipelines. A lightweight runtime featuring a micro-container design is performance-optimized for high-throughput NGS analysis, and includes powerful data management features.

The Chromium™ Software Suite

Linking data, developers and discovery

Changing the Definition of Sequencing™10x Genomics

13 14

The Loupe™ family of visualization applications brings clarity to the novel biological insights made possible by the Chromium System.

For genomes and exomes: fully haplotype-enabled genome browsing and structural variant visualization. For single cell transcriptomics: dimensionality reduction, clustering, and isolation of cell types and phases. Loupe applications feature fluid, modern user interfaces, run on Windows and Mac, and work with files produced by the Long Ranger and Cell Ranger pipelines.

The Loupe structural variant view lists the calls and candidates produced by the Long Ranger pipelines, and uses changes and gaps in color intensity to represent barcode-based evidence of structural rearrangements.

The Loupe haplotype view displays multi-megabase phase blocks and structural variant calls. Above, an example of compound heterozygosity is shown where the variants involved are fully contained within large, contiguous phase blocks.

The Loupe single cell analysis view features an array of techniques for dimensionality reduction and clustering which can be applied to gain insight into a variety of single cell experiment types.

10x Genomics

15 16

Changing the Definition of Sequencing™

10x Genomics, Inc.

About us10x Genomics meets the critical need for long range, structural and cellular information, with an innovative system that transforms the capability of existing short-read sequencers. Our Chromium™ System supports comprehensive genomics and high-throughput single cell transcriptomics. It enables researchers to discover previously inaccessible genomic information at unprecedented scale, including phased structural variants, phased single nucleotide variants, and dynamic gene expression of individual cells—while leveraging their existing sequencing systems and workflows.

Contact Us

Pleasanton, CA10x Genomics, Inc. 7068 Koll Center Parkway, Suite 401 Pleasanton, CA 94566

+1 925 401 7300 +1 800 709 1208 info@10xgenomics.com

San Francisco, CA10x Genomics, Inc. 160 Spear Street, Suite 1130 San Francisco, CA 94105

+1 925 401 7300 +1 800 709 1208 info@10xgenomics.com

© 2016 10x Genomics, Inc. All rights reserved. Duplication and/or reproduction of all or any portion of this document without the express written consent of 10x Genomics, Inc., is strictly forbidden. Nothing contained herein shall constitute any warranty, express or implied, as to the performance of any products described herein. Any and all warranties applicable to any products are set forth in the applicable terms and conditions of sale accompanying the purchase of such product. 10x Genomics provides no warranty and hereby disclaims any and all warranties as to the use of any third party products or protocols described herein. The use of products described herein is subject to certain restrictions as set forth in the applicable terms and conditions of sale accompanying the purchase of such product. “10x”, “10x Genomics”, “Changing the Definition of Sequencing”, “Chromium”, “GemCode”, “Loupe”,

“Long Ranger”, “Cell Ranger”, “Lariat” and “Supernova” are trademarks of 10x Genomics, Inc. All other trademarks are the property of their respective owners. All products and services described herein are intended FOR RESEARCH USE ONLY and NOT FOR USE IN DIAGNOSTIC PROCEDURES.

10xGenomics.com

10x Genomics

17

Changing the Definition of Sequencing™10xGenomics.com