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10x Genomics Single-Cell Sequencing

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CD genomics is now offering single cell analysis utilizing various solutions of the 10X Genomics Chromium system, allowing the profiling of rare or heterogeneous populations of cells.

The Introduction of 10x Sequencing

Powerful approaches have become more accessible that allow the profiling of rare or heterogeneous populations of cells. The averaging that occurs in Conventional 'bulk' methods of sequencing does not allow the direct assessment of the fundamental biological unit—the cell—or the individual nuclei that package the genome. CD genomics provides access to the genomic and genetic analysis at single cell level using 10x genomics system and its engineered reagent delivery method. The droplet-based platforms Chromium 10x System, powered by GemCode Technology, enables the biomedical researchers and clinicians to make important new discoveries using this powerful approach as the technologies and tools needed for conducting single cell studies. Moreover, the droplet-based platforms, maximizing the throughput of cells and meanwhile downscaling the reactions to nanoliter volumes, has been shown to improve detection sensitivity and quantitative accuracy. The droplet-based methods hold advantages, allowing direct analysis of rare cell types or primary cells for which there may be insufficient material for conventional bulk sequencing protocols. It is also ideal to profile interesting subpopulations of cells from a larger heterogeneous population, for example, malignant tumor cells within a tumor mass, or hyper-responsive immune cells within a seemingly homogeneous group. It can even reveal entirely new cell types. 10X genomics also permits to study cellular states in scenarios such as embryonal development, cancer, myoblast and lung epithelium differentiation, and lymphocyte fate diversification.

Encapsulate your sample into hundreds to tens of thousands of uniquely addressable partitions in minutes, each containing an identifying barcode for downstream analysis. Each Gel Bead, infused with millions of barcoded oligonucleotides, is mixed with a sample, which can be high molecular weight (HMW) DNA, individual cells, nuclei, or Cell Beads. 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 (Figure 1). Barcoded products are pooled for downstream reactions to create short-read sequencer compatible libraries. After sequencing, the resulting barcoded short read sequences are fed into turnkey analysis pipelines that use the barcode information to map reads back to their original HMW DNA, single cell, or single nucleus of origin.

Schematic overview of the fragment of a final 10x Chromium Genome library
Schematic overview of the fragment of a final 10x Chromium Genome library

Chromium™ Single Cell Gene Expression Solution

Maximizing the throughput of cells is key to identify the cell types from complex tissues. The 10X Genomics Chromium Single Cell Gene Expression Solution can be used to perform transcriptome measurement in individual cells with sequencing large numbers of single cells can recapitulate bulk transcriptome complexity. CD Genomics is dedicated to providing high-quality single cell transcriptome profiling service using the 10X Genomics Chromium Single Cell Gene Expression Solution, along with turnkey software tools, which allows the creation of high complexity libraries from single cells to maximize insight from any sample type. The Cell Ranger analysis pipelines perform primary analysis and visualization. The Cell Ranger analysis pipelines perform standard analysis steps such as demultiplexing, alignment, and gene counting. Cell Ranger leverages the10x Barcodes to generate expression data with single-cell resolution. This data type enables applications including cell clustering, cell type classification, and differential gene expression at a scale of hundreds to tens of thousands of cells.

The Chromium™ System enables single cell transcriptional profiling of up to tens of thousands of cells (typically 1000-6000), by partitioning cells into nanoliter-scale GEMs, where all generated cDNA share a common 10x Barcode is used to associate individual reads back to the individual partitions. Incubation of the GEMs produces barcoded, full-length cDNA from poly-adenylated mRNA Single Cell 3' Library comprises standard Illumina paired-end constructs which begin and end with P5 and P7. 16 bp 10x Barcode and 12 bp UMI are encoded in Read 1, as the first base pairs of the library insert. Sample index sequences are incorporated as the i7 index read.

The Chromium™ cell partitioning workflow for scRNA sequencing
The Chromium™ cell partitioning workflow for scRNA sequencing

10x Genomics Single Cell Protocols require a suspension of viable single cell or single nucleus as input. Researchers must submit a dissociated suspension of live cells. To obtain high-quality data, when it is crucial to maximize viability and minimize the presence of nuclear aggregates, dead cells, cellular debris, cytoplasmic nucleic acids, and potential inhibitors of reverse transcription. Accurate cell counting is critical to application success. The single cell suspension between 700-1200 cells/ul is recommended for optimal target cell recovery. It is recommended that the cell suspension should be counted 3-4 times and the standard deviation between all counts should be < 25%.

  • Feature barcoding: cell surface barcoding

Feature barcoding technology enables multiplxing and doublet detection and/or identifying cell-surface protein isoforms, detecting protein for low abundance transcripts, and further increasing phenotypic specificity.

  • Feature barcoding: CRISPR screening

Implementing the CRISPR screening to conduct high-throughput and scalable functional genetic screens in hundreds to tens of thousands of single cells simultaneously.

Chromium™ Single Cell Immune Profiling Solution

The Chromium Single Cell Immune Profiling Solution is a comprehensive approach to understand the adaptive immune system of hundreds to tens of thousands of T and B cells in human or mouse with single cell resolution. CD genomics offers streamlined workflows that allow you to go from cells suspension to library prep, immune sequencing, and software analysis, enabling the assembly and annotation of full- length V(D)J segments and simultaneously assessment of TCR, Ig, and 5' gene expression in the same cell.

Single cell suspensions loaded onto the system are partitioned into GEMs, where transcripts are tagged with cells 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. The protocol produces Chromium Single Cell V(D)J libraries ready for Illumina sequencing.

For V(D)J enriched libraries, Read 1 encodes the 16 bp 10x™ Barcode, 10 bp UMI, and 13 bp Switch Oligo, as well as the 5' end of an enriched transcript. For 5' gene expression libraries, Read 1 encodes the 16 bp 10x Barcode and 10 bp UMI. Due to Enzymatic Fragmentation, for both libraries Read 2 encodes a random internal fragment of the corresponding insert. Sample index sequences are incorporated as the i7 index read. A schematic of the final library constructs is shown below.

Chromium Single Cell V(D)J Enriched Library
Chromium Single Cell V(D)J Enriched Library

5' Gene Expression Library Structure5' Gene Expression Library Structure

The Chromium™ linked-reads Sequencing Solution

The Chromium Genome Sequencing Solution enables molecular barcodes to tag reads that come from the same long DNA fragment, which provides the long-range information missing from standard approaches. As a result, we are able to link the short reads together by using a unique barcode to every short read generated from an individual molecule. Linked-reads can help access NGS dead zones, find more structural variants, resolve haplotypes and easily de novo genome assemblies. 

For whole genome analyses, starting the process with high molecular weight (HMW) genomic DNA, sequencing-ready libraries of copies of unique barcoded gDNA is created. For whole exome analyses, by adding the Agilent SureSelectXT Human All Exon V6 Reagent Kit, the Target Enrichment libraries provide optimal exome application performance as well. Optimal performance has been characterized on input gDNA with a mean length greater than 65 kb, and this protocol outlines the extraction of HMW gDNA with optimal size from live cells.

The Chromium™ Single Cell Copy Number Profiling

CD genomics has the ability to sequence libraries from the DNA of single cells and analyze the genomic data, accurately detecting single cell CNV events.

Building on the 10x GEMs technology, The Chromium Single Cell CNV Solution is able to profile hundreds to thousands of cells. DNA from single cells is barcoded within the Cell Bead Gel Bead partitions and the barcoded fragments are then pooled for library production. The barcoded library fragments can be easily traced back to the cells from which they originated using downstream bioinformatics tools. It provides a comprehensive, scalable approach to determine genome heterogeneity and map clonal evolution by profiling hundreds to thousands of cells in a single sample. It is easier than ever to study complex pathogenesis, including cancer progression and genetic disorders, at unprecedented scale and resolution. Single cell CNV events will be called at around 2Mb resolution, but on clusters of cells, CNV events can be detected down to hundreds of Kb resolution.

The Chromium™ ATAC Sequencing

The 10x Chromium™ System designed single cell ATAC (Assay for Transposase Accessible Chromatin) solution to help understand the regulatory landscape of the genome. also enables understanding of epigenetic and regulatory variation across tens of thousands of cells with the Chromium Single Cell Assay for Transposase Accessible Chromatin (ATAC) Solution. Starting with hundreds to tens of thousands of nuclei, transposase enzyme is used to preferentially tag accessible DNA regions with sequencing adaptors. Transposed DNA fragments from individual nuclei are distinguished with one of ~750,000 possible 10x Barcodes. It profiles 500 to 10,000 nuclei per chanel and interrogate open chromatin profiles of individual nuclei.

Deliverables

  • The original sequencing data
  • Experimental results
  • Data analysis report
  • Details in Next Generation Sequencing for your writing (customization)

If you have additional requirements or questions, please feel free to contact us.

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Tel: 1-631-275-3058 (USA)
       44-208-144-6005 (Europe)
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Email: info@cd-genomics.com