Space Ranger6.4 (latest), printed on 05/24/2023
Goal: To evaluate the expression profiles of known gene markers of interest in the context of tissue morphology.
This tutorial will showcase how to obtain the spatial expression patterns of known gene markers associated with distinct cell types using a preloaded mouse brain dataset in Loupe Browser.
Table of Contents
- Explore tissue image
- Filter gene markers
- Neuronal markers
- Oligodendrocyte markers
- Microglia markers
- Gene expression independent spatial enrichment (Moran's I)
- Installed Loupe Browser on either macOS or Windows. Instructions on the Installation page.
- Familiarity with Loupe Browser navigation. Learn more in theNavigation Tutorial.
- Access the tutorial dataset. Instructions here.
Explore tissue image
To effectively evaluate gene expression data in the context of the tissue image,it is helpful to understand the underlying morphology that can beuncovered from the tissue image. This can be achieved by making the spots transparent using the Spot Opacity slider located in the top left hand corner and moving it all the way to the left.
Using a combination of zoom and mouse dragging functionality navigatethe image, evaluate the morphology, and identify landmarks of interest. You can zoom using either the mouse or the zoom slider. To revert back to default view click. Forexample, in this image, the curvature of the hippocampus is highlighted in blue staining. The lateral ventricle is highlighted by a blue arrow.
Filter gene markers
Loupe Browser provides the capability to filter the expression for specific gene or list of genes using the Active Feature List. Click on the mode selector to select Gene/Feature Expression and click on to start typing.
To visualize the gene expression, make the spots opaque using the Spot Opacity slider. First we will explore the neuronal cell types using some well known markers. Type Map2, which is responsible for microtubule protein that provides the structural support to neurons, in the text box. The auto-complete functionality helps tofind genes of interest faster. Add the gene into the Active Feature List using any one of these methods:
- Click on the name from the list
- For Windows press or for macOS press
The tissue image is then updated to color-code spots based on thelevel of Map2 within that spot. High expression of the gene is indicated by adarker spot. As expected high Map2 expression is spread across the tissue as it isa general mature neuronal marker. Some spots appear out of the range of the color scale, in this case, light gray and correspond to spots that show no expression of the selected gene.
Hovering over a gene name reveals two icons, an information iconand a trash icon. Clicking loads the Ensembl reference page for that gene as a new tab in web browser. Click to remove that gene from the current list. The color scale option has been updated from default using the color palette in the bottom-right corner of the browser.
Next continue with these additional neuronal markers:
- Rbfox3 which is shown to be important for neurogenesis and synaptogenesis especially in the subgranular layer of the hippocampus
- Slc17a7 encodes a vesicle-bound, sodium-dependent transporter specifically found in glutamatergic neurons
- Gad1 encodes glatamic acid decarboxylase 67 which is crucial for gamma-aminobutyric acid (GABA) neurotransmission in neurons
These markers give clear indication of the distinction in spatial expression. Rbfox3 and Slc17a7 are more prominently present in excitatory neurons in hippocampus and cerebral cortex while Gad1 has a heavier presence in the inhibitory neurons of the thalamus region.
Thus far, we have been examining the expression pattern for each gene. If you click the actively selected gene and deselect it, the resulting color signature represents a combination of expression levels across all the genes in the list. This allows you to visualize a set of genes associated with a cell type more confidently.
Oligodendrocytes produce myelin sheaths that surround the axons of neurons. We will use two markers to identify this cell type:
- Mbp which encodes the myelin basic protein which is a major constituent of the myelin sheath
- Mog which is a glycoprotein uniquely expressed on the surface of oligodendrocytes
As expected the strong expression of these markers coincide with anatomical locations of the major white matter tracks including corpus callosum, fimbria, cerebral peduncle and optic track.
However not all genes show definite patterns in spatial expression. As an example, lets look at some markers for microglia cells which are the resident immune cells:
- Cx3cr1 is a chemokine receptor highly expressed in microglia
- Tmem119 which is a cell-surface protein recently characterized as a microglial marker
You can export this list for future reference by clicking the three vertical dots and choosing Export Lists option which downloads a file in CSV format to a location of your choice. The option to import a feature list is discussed in Explore Spatial Clusters tutorial.
Gene expression independent spatial enrichment (Moran's I)
Another metric available to examine the spatial distribution of genes is by using the spatial correlation coefficient Moran's I which ranges from -1 (perfectly dispersed) to 1 (perfectly enriched). This metric is available for datasets analyzed using Space Ranger 1.3 or later.
Click on in the bottom left corner to access the spatial enrichment table. The features displayed in this table are filtered by Moran's I ≥0.05 and p-value ≤0.05 (p-value adjustment is performed using the Benjamini-Hochberg Procedure) and rounded to two decimal places to highlight the most spatially correlated features. The full list of Moran's I values for any dataset is captured in spatial_enrichment.csv which is generated in the
outs/ folder after spaceranger count run.
The Spatial Enrichment table can be sorted by Moran's I value, UMI count, and number of Spots for which at least one valid UMI is detected for the feature. The list of features can also be filtered by a specific Moran's I value range using the Filter Table . The filtered and sorted list can be exported in CSV format by clicking. Clicking on next to column headers provides a pop box with information. Note that you can resize the table to display more rows using your cursor to drag the top border of the panel up. Clicking on the feature name pops a window with the option to add to Active Feature List.
We will now examine two set of genes for examples to understand spatiality. Note the values visualized correspond to the Log2 value of the Feature Max attribute.
For the first set we choose genes Ttr and Defb11. Note that you can either use the search option in the Active Feature List or click on the feature name in the spatial enrichment table to add it to the list.
Ttr is detected across many spots (2674) and the Moran's I value is 0.81. The spatial pattern looks spread out with high expression in lateral and third ventricles. On the other hand Defb11 is detected in smaller number of spots (61), has Moran's I of 0.52 and the spatial pattern is localized to only the lateral and third ventricles. This is consistent with both the gene function and cell type association.
Ttr or Transthyretin encodes a carrier protein for thyroxine and retinol in cerebrospinal fluid (CSF) and is highly expressed in choroid plexus epithelial cells. However, it's RNA has been detected in cortex, hippocampus and thalamus. Defb11 or Defensin beta 11 encodes a antimicrobial peptide and is expressed in choroid plexus epithelial cells. Since it is in involved in innate immunity, the baseline expression in low.
For the second example, we will look at oligodendrocyte genes Mbp, Mobp and Mog.
A decreasing trend of highly expressed spots is visible moving from left to right in the image above. The spatial enrichment values in the table confirm the visual observation.
|Name||Moran's I||UMI Count||Spots|
This is consistent with cell type association. Mbp and Mobp are associated with myelin forming and mature oligodendrocytes. They are also expressed in excitatory, inhibitory and cholinergic neurons in hindbrain as well as afferent nuclei of cranial nerves. Mog on the other hand is only associated with myelin forming and mature oligodendrocytes and hence show lesser degree of spatial clustering.
Using the methods shows in the tutorial, you can map known gene markers to spots, overlay that information on top of the tissue image, and use that to verify that the expected gene patterns are present in their respective brain tissue types.
How do you identify a marker gene? ›
Marker genes, defined as genes that are expressed primarily in a single-cell type, can be identified from the single-cell transcriptome; however, such data are not always available for the many uses of marker genes, such as deconvolution of bulk tissue.What are the best practices for flow sorting cells for 10x Genomics assays? ›
Use a larger flow nozzle (such as a 100um nozzle, if using a sorter with a nozzle), or use lower pressure when sorting your cells. Using lower pressure during the sorting process will help preserve cell health and viability. In most circumstances, it is better to sort on a lower flow rate to maintain cell health.What does 10x mean in Genomics? ›
10x Genomics is a microfluidics-based method of single-cell RNA sequencing. The technique makes use of the Chromium system, a device that enables single-cell sequencing with their Next GEM technology.How does 10x Genomics sequencing work? ›
10x Genomics' single cell RNA-seq (scRNA-seq) technology, the Chromium Single Cell 3' solution, allows you to analyze transcriptomes on a cell-by-cell basis through the use of microfluidic partitioning to capture single cells and prepare barcoded, next-generation sequencing (NGS) cDNA libraries.What are the 3 types of markers? ›
Because normal DNA or protein molecules are used to score the genetic material, molecular markers are phenotypically neutral. This is a significant advantage compared to traditional phenotypic markers. The three most common types of markers used today are RFLP, RAPD and isozymes.What are genetic evaluation markers? ›
A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be observed.What does 10X value mean? ›
10x means to maximize and expand your results ten times over, rather than just by 10%.What does 10X increase mean? ›
What Is The 10X Rule? Put very simply, the 10X rule is taking any goal you've set for your company or sales team, and multiplying it by 10. So if a goal is to increase revenue by 5%, using the 10X rule, you'd increase that goal to 50%.What is the gene expression of a cell? ›
Gene expression is a tightly regulated process that allows a cell to respond to its changing environment. It acts as both an on/off switch to control when proteins are made and also a volume control that increases or decreases the amount of proteins made.How many cells needed for 10x Genomics? ›
The optimal cell concentration for a 10X Genomics single cell RNA sequencing experiment is 400-1200 cells/µL in a minimal volume of 30-100 µL.
Is 10x Genomics strand specific? ›
Answer: Yes, 10x Single Cell solutions are strand-specific. Cell Ranger 'Count' counts sense-strand reads only.What tips are compatible with 10x Genomics? ›
10x Genomics recommends using only validated emulsion-safe pipette tips for all Single Cell protocols. Rainin pipette tips have been extensively validated by 10x Genomics and are highly recommended for all single cell assays.Which technique is the best one to study levels of gene expression in multiple genes and tissues? ›
The microarrays are used to determine expression levels across a large number of genes or to perform genotyping across different regions of a genome.
The maximum cell size we have tested in-house is 30 µm. Larger cells may clog the microfluidic channels of 10x Genomics chips.Why are genetic markers important? ›
Genetic markers can help link an inherited disease with the responsible gene. DNA segments close to each other on a chromosome tend to be inherited together. Genetic markers are used to track the inheritance of a nearby gene that has not yet been identified, but whose approximate location is known.What are examples of genetic markers? ›
Examples of genetic markers are single polymorphism nucleotides (SNPs), restriction fragment length polymorphisms (RFLPs), variable number of tandem repeats (VNTRs), microsatellites, and copy number variants (CNVs). Genetic markers may or may not have a known function.What is the difference between a marker and a gene? ›
Selectable markers are sequences that can be used to distinguish between transformed and non-transformed genes. Reporter genes are test sequences whose expression can be quantified. They act as markers for transformed genes by providing resistance against toxic substances.What are the two types of marker genes? ›
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. In particular, there are two sub-types of these marker genes: a selectable marker and a marker for screening.What are the most common genetic markers? ›
In the human genome, the most common types of markers are SNPs, STRs, and indels. SNPs affect only one of the basic building blocks—adenine (A), guanine (G), thymine (T), or cytosine (C)—in a DNA segment.How many genetic markers are there? ›
We have constructed a genetic linkage map of human chromosome 16 based on 46 DNA markers that detect restriction fragment length polymorphisms.
How many percent is 10x? ›
1000% of something is 10 times it. 1000% more than the same thing is 11 times it.What is the return of 10x in 10 years? ›
10x in 10 years is 26% annualized. If you have 1 stock out of 10 that did this, it would be responsible for about 1/4th the total return you got in the last 10 years.What does 10x mean in VC? ›
What is a 10x potential? In simple terms: If you want to have 10 million dollars invested in your company, you need to show the VCs a way to return a minimum of 100 million dollars in 5 to 10 years. It is a minimum of 10 times the invested capital returned.How do you calculate 10X growth? ›
Obviously, the way to calculate a return multiple is to divide the amount returned from an investment by the dollars invested. If I invested $10M in a company and got back $100M, that's a 10X return.What does 10X and 100X mean? ›
read it till end. Let me decode it ! In 10X or 100X, X is the initial money or worth or valuation or order or revenue at the time T e.g. today or this quarter or this year. And 100X is 100 times of today's money or worth or valuation or order or revenue in a specified time period.What is the difference between 10% and 10X? ›
Incremental change (10%) is constant, while exponential change (10X) has an increasing rate. The incremental is linear and additive, while the exponential is non-linear and multiplicative. While the incremental is about 10% improvements, the exponential is about 10X acceleration.What are 10X ideas? ›
The 10X Rule says that 1) you should set targets for yourself that are 10X greater than what you believe you can achieve and 2) you should take actions that are 10X greater than what you believe are necessary to achieve your goals.What does scale of 1 to 10 mean? ›
A drawing at a scale of 1:10 means that the object is 10 times smaller than in real life scale 1:1. You could also say, 1 unit in the drawing is equal to 10 units in real life. As the numbers in the scale get bigger, i.e. 1:50 – 1:200, the elements in the drawing actually get smaller.How do you interpret a number scale? ›
The number scale is expressed as a ratio like 1 : 50. This simply means that 1 unit on the map represents 50 units on the ground. So 1 cm on the map will represent 50 cm on the ground, or 1 m on the map will represent 50 m on the ground.What can gene expression tell us? ›
Gene expression be thought of as an “on/off switch” to control when and where RNA molecules and proteins are made and as a “volume control” to determine how much of those products are made.
How do you explain gene expression? ›
The process by which a gene gets turned on in a cell to make RNA and proteins. Gene expression may be measured by looking at the RNA, or the protein made from the RNA, or what the protein does in a cell.How do you measure gene expression? ›
Gene expression measurement is usually achieved by quantifying levels of the gene product, which is often a protein. Two common techniques used for protein quantification include Western blotting and enzyme-linked immunosorbent assay or ELISA.What is the minimum number of cells for 10X? ›
With 10x Genomics, you can aim to target between 3,000 and 10,000 cells per sample. Its minimal target of 3,000 cells can make the starting experiment relatively expensive.What is the dropout rate of 10X Genomics? ›
Dropout events in scRNA-seq can result in many genes undetected and an excess of expression value of zero, leading to challenges in differential expression analysis , . The average dropout ratios of majority genes in 10X were 1.3–1.4 folds those in Smart-seq2 for all samples tested (Figure 5A, Figure S7A).What is the cell capture rate of 10X? ›
10X Chromium Single Cell Features:
Captures 100-20,000+ cells per sample and up to 320,000 cells (or ~ 1 million cells with multiplexing) per run in ~20 minutes. Recovers up to ~65% of cells (typically 50%). Low doublet rate (~0.9% per 1,000 cells).
The 10x Genomics Multiome product is compatible with all Illumina® platforms, but we recommend sequencing on NextSeq™ 550/1000/2000 and NovaSeq™ 6000 to be able to sequence multiple samples per run (see table below).Who is 10x Genomics owned by? ›
Serge Saxonov co-founded 10x Genomics in 2012. As the CEO, he defined the 10x vision and strategy, contributed to core inventions, and has led the company since inception.What is the difference between drop seq and 10x Genomics? ›
- In contrast to Drop-seq, where solid beads are used for RNA capture, 10X uses soft hydrogels containing oligos. These enable “single Poisson loading” leading to capture of >60% of input cells.How are cell markers identified? ›
First, more and more cell markers of diverse cells (such as some new/rare cell types) will be identified by single-cell RNA sequencing technology.What is an example of a marker gene? ›
Examples of genetic markers are single polymorphism nucleotides (SNPs), restriction fragment length polymorphisms (RFLPs), variable number of tandem repeats (VNTRs), microsatellites, and copy number variants (CNVs). Genetic markers may or may not have a known function.
What is one example of marker gene? ›
Example: Kanamycin resistant gene.What are identifying markers? ›
Evidence identification markers are items that are used to mark and illustrate items of evidence at a crime scene. These are commercial products like plastic alpha-numeric stands, markers, cones, and flags. The stands and label products come in various sizes, colors and shapes.Where are marker genes found? ›
A genetic marker is a DNA sequence with a known physical location on a chromosome. Genetic markers can help link an inherited disease with the responsible gene. DNA segments close to each other on a chromosome tend to be inherited together.How are cell markers identified which markers are used to identify the stem cells? ›
Stem cell markers are genes and their protein products used by scientists to isolate and identify stem cells. Besides, stem cells can also be identified by functional assays which are considered the gold standard for the identification and therapeutic purposes.What is the most commonly used marker gene? ›
Background: The 16s rRNA gene is so far the most widely used marker for taxonomical classification and separation of prokaryotes.What are the most common marker genes? ›
The most widely used selectable marker is bar gene isolated from Streptomyces hygroscopicus. It codes for phosphinothricin acetyl transferase (PAT) proteins of 183 amino acids and shows 85% DNA sequence homology with another marker gene pat isolated from Streptomyces viridochromogenes.What is the importance of marker genes in vectors? ›
Selectable markers are often antibiotic resistance genes. They help in artificial selection. They help in identifying transformants and selectively allowing their growth whereas eliminating non-transformants in cloning vector.What is an example of a cell marker? ›
Cell surface markers are special proteins expressed on the surface of cells or carbohydrates attached to the cell membrane that often conveniently serve as markers of specific cell types. For example, T cell and B cell surface markers identify their lineage and stage in the differentiation process (Figure 1).What is the purpose of markers? ›
A marker is an object which is used to show the position of something, or is used to help someone remember something.Are genetic markers accurate? ›
The accuracy of genetic tests to detect mutated genes varies, depending on the condition being tested for and whether or not the gene mutation was previously identified in a family member. Even if you don't have the mutated gene, that doesn't necessarily mean you'll never get the disease.
What is the function of a marker? ›
Markers are used as signposts (or landmarks) in the construction of DNA and genome maps. Markers can also be used to track inheritance of traits or disease risk in families.