November 19, 2018

10X Genomics single cell and linked-reads

A GEM is a “Gel bead in EMulsion” droplet that encapsulates each micro-reaction within the Chromium System. Here we show a Single Cell GEM with a single T cell, reagents, and barcoded gel bead all partitioned within a single oil droplet.

The 10X Genomics Chromium System, provides a reagent delivery method that enables thousands of micro-reactions in parallel, each containing an identifying barcode such that all sequencing library fragments produced within a partition share a common barcode.

Gel Beads, containing barcoded oligonucleotides, are mixed with a sample, which can be high molecular weight (HMW) DNA, cells, or nuclei. Gel Beads and samples are then added to an oil-surfactant solution to create Gel Beads in EMulsion (GEMs), which act as individual reaction vesicles in which the Gel Beads are dissolved and the sample is barcoded. Barcoded products are pooled for downstream reactions to create short-read sequencer compatible libraries. After sequencing, the resulting barcoded short read sequences are analyzed with bioinformatics that use the barcode information to map reads back to their original HMW DNA, single cell, or single nucleus of origin.

 

 

DNA partitioning for linked read sequencing

 

LINKED READS GENOMICS

High molecular weight (HMW) DNA partitioning → linked reads

For whole genome or exome analyses, the Chromium Controller allows researchers to create sequencing-ready libraries with >1,000,000 unique barcodes from ~1 ng of HMW genomic DNA. Long input DNA molecules are barcoded, and are then sequenced in bulk to produce a unique data-type known as Linked-Reads.

Genome and Exome Sequencing
Uses the power of Linked-Reads to fully resolve genic phasing, reveal structural variation and detect variants in previously inaccessible and complex regions of the genome. Allows phased calling of the full spectrum of human genetic variations, including SNPs, small indels, and complex structural variants.

de novo Assembly
The Chromium de novo Assembly Solution opens the door to low-cost, every day diploid genome assemblies. With its simple workflow, low DNA input requirements, and automated data analysis, the Chromium de novo Assembly Solution enables true diploid genome assembly like never before. The long range information encoded in barcoded Linked-Reads is leveraged by innovative bioinformatics pipelines to assemble sequences over long genomic distances, including across repetitive regions. The precise assembly of Linked-Reads leverages heterozygous loci to resolve individual haplotypes, enabling diploid de novo assembly.

 

 

Partitioning workflow for single cell transcriptional profiling

 

SINGLE CELL TRANSCRIPTOMICS

Partitioning of Individual cells.

The Chromium System also enables single cell transcriptional RNA profiling of hundreds to tens of thousands of individual single cells. Single cell suspensions loaded onto the system are partitioned into GEMs, where transcripts are tagged with cell-specific barcodes. The barcoded cDNA is then pooled for downstream processing and library preparation. For immune repertoire profiling, the cDNA undergoes targeted enrichment for T- or B-cell receptor transcripts prior to library preparation. After sequencing, downstream bioinformatics tools use the cellular barcodes to group transcripts that originated from the same cell, revealing the transcriptome and/or the full-length T- or B-cell receptor sequences of each individual cell.

Single Cell Gene Expression
High-throughput, single cell gene expression measurements that enable discovery of gene expression dynamics and molecular profiling of individual cells and cell types.

Gene Expression & cell-surface protein (using Feature Barcoding technology)
Seamlessly measure both gene- and cell surface protein-expression in the same cell to identify protein isoforms, detect protein for low abundance transcripts, and further increase phenotypic specificity.

Gene Expression CRISPR screening (using Feature Barcoding technology)
Identify perturbations of cellular transcripts mediated by hundreds of different CRISPR sgRNAs in one experiment.

Single Cell Immune Profiling
Tool for profiling full-length paired V(D)J transcripts from hundreds to tens of thousands of lymphocytes. The new solution enables assembly of full-length V(D)J sequences on a cell-by-cell basis, providing high resolution insights into the adaptive immune system.

Single Cell Immune Profiling & cell-surface protein (using Feature Barcoding technology)
Seamlessly combine cell surface protein expression measurements along with immune repertoire and gene expression measurements from the same single cell.

Single Cell Immune Profiling & antigen specificity (using Feature Barcoding technology)
Determine antigen specificity of single T cells with Feature Barcode peptide-MHC multimers to study the dynamic interactions between lymphocytes and antigens. Unveil a new understanding of the adaptive immune response in hundreds to tens of thousands of lymphocytes.

 

SINGLE CELL GENOMICS

Single Cell CNV
Revealing genome heterogeneity, understanding clonal evolution, characterize neuronal mosaicism at the single cell level, etc.

 

SINGLE CELL EPIGENOMICS

Single Cell ATAC
Provides genome-wide chromatin accessibility information for tens of thousands of nuclei. Elucidate gene regulatory mechanisms to understand epigenetic heterogeneity.