Week 13 (Single Cell) Flashcards
1
Q
ominis cellula e cellula
A
all cells arise from cells
2
Q
what are the epochs of genome research?
A
- The Human Genome Project
- ENCODE
- GTEx Project
- Single Cell
3
Q
there are A LOT of variants. what is the problem with having a lot of variants?
A
we need to find a way to know the mechanistic features of these variants, can use single cell technology
4
Q
What are the three themes of single cell research?
A
- Theme 1: discovery of human variation
- Theme 2: from variant to function
- Theme 3: precision health
5
Q
what is the basic unit of life?
A
the cell
6
Q
who first define dead cells in plants?
A
Robert Hooke
7
Q
who was the first to define live cells?
A
Leeuwenhoek
8
Q
there are roughly ____ cells in the human body
A
37 trillion
9
Q
what are the oldest cells in the human body?
A
heart cells (can live up to 40 years)
10
Q
what is the largest cell in the human body?
A
oocyte
11
Q
which cells have the shortest life duration?
A
red blood cells
12
Q
what is a majority of the cell made up of?
A
water (70%)
13
Q
how many cell types are known?
A
about 200
14
Q
what are stem cells?
A
cells that can differentiate into any cell types
15
Q
!! what is a cell type? !!
A
a classification used to describe cells that share a common structural, functional, and molecular characteristics.
cells of the same type typically perform similar roles within an organism and express distinct sets of gene that define their identity.
16
Q
what are the 4 main ways cell types have been differentiated?
A
- morphology
- gene expression profile
- function
- location
17
Q
cell type: morphology
A
cells often have characteristic shapes, sizes, and structural features
18
Q
cell type: gene expression profile
A
each cell type has a unique pattern of gene activity that defines its function and identity
19
Q
cell type: function
A
different cell types are specialized for specific biological tasks, such as muscle contraction, immune defense, or neurotransmission
20
Q
cell type: location
A
cell types are often found in specific tissues or organs
21
Q
what are the cell/nuclei isolation points?
A
- tissue source will dictate isolation protocol
- cell liberation conditions highly variable by tissue source
- cell lysis conditions holy variable by tissue source for nuclei preparation
22
Q
what dictates an isolation protocol when collecting a single cell or nuclei for sample preparation?
A
tissue source
23
Q
what does tissue source dictate how a sample is collected and prepared?
A
some cells are going to be more difficult to separate from extracellular proteins than others
24
Q
what is used when we need to isolate a cell vs isolate a nuclei?
A
- cell = proteases
- nuclei = detergents
25
why do you need to use a detergent to isolate nuclei?
detergents are used to isolate nuclei by disrupting the cell and cytoplasmic membranes, allowing for the release and purification of nuclei
26
why do we stain cells or nuclei?
to know what you have and the quality of the sample
27
what is the red vs the green?
- red = dead
- green = alive
28
what does this image tell you about the sample?
something went wrong because the nuclei should be red, meaning they should be dead
29
what is nuclear blebbing?
herniations of the nucleus that occur in diseased nuclei that cause nuclear rupture leading to cellular dysfunction
30
is nuclear blebbing good or bad?
BAD, you do not want this
31
define apoptosis
the death of cells which occurs as a normal and controlled part of an organism's growth and development
32
what is bulk RNA sequencing?
analyze expression change in a mixture of cells
33
what is single cell RNA sequencing?
analyze gene expression in a single cell or nuclei
34
what is the difference between Bulk RNA sequencing and single cell RNA sequencing?
Bulk RNA sequencing analyzes RNA from a pooled population of cells, providing an average gene expression profile across the entire sample. Single-cell RNA sequencing, on the other hand, examines RNA from individual cells, revealing gene expression variations within a heterogeneous population. Single cell allows for multiple and specific genes in the cells to be studied.
35
what are key advancements made in single-cell RNAseq?
1. integrated flow cell circuits
2. nanodroplets
3. in situ barcoding
36
what drives scRNAseq technology adoption?
- cost
- ease of the technique
- data robustness
- experimental objectives
- personnel bias
37
major steps to sc/snRNAseq data generation:
1. lipid encapsulation of beads, cells, and transcription enzyme mix
2. cell lysis and mRNA binding to the capture beads
3. cDNA synthesis with reverse transcriptase
4. pooling all multi-barcoded cDNA and sequencing
38
what is the most common single cell/nuclei technology?
10x genomics
39
what is the input and output of the single cell digital gene expression technology?
- input: single cells in suspension + 10x gel beads and reagents
- output: digital gene expression profiles for every partitioned cell
40
what is a reverse transcriptase?
an enzyme that converts RNA into DNA
41
what enzyme was discovered when scientists were working with Rous sarcoma virus (RSV)?
reverse transcriptase
42
why does reverse transcriptase have a high error rate?
it has no proofreading ability
43
reverse transcriptase has a high error rate. Is this good or bad?
- good for the virus because it can escape immune reaction
- this may be okay or bad for cDNA depending on how high the error rate is
44
how many cycles are recommended when amplifying cDNA?
12 cycles
45
why are only 12 cycles recommended when amplifying cDNA?
if you do too many cycles you can get bias in your data
46
what are the molecular techniques that allow s to achieve single cell resolution?
- cell barcode
- UMI
- sample index
- (adapters)
47
make a diagram that represents the molecular techniques that allow is to achieve single cell resolution
(label p5 and p7, cell barcodes, UMI, and sample index)
48
what is the molecular technique that allows us to achieve cell resolution?
(in situ) barcoding!!
49
why do we need a cell barcode?
to know the origin of the individual cell
50
why do we need a UMI?
UMIs are short, random DNA sequences that are added to the cDNA molecules during library preparation. Each original molecule receives a unique UMI, allowing researchers to track the origin of each amplified sequence
51
why do we need a sample index?
to know which sample the read came from
52
what is the purpose of P5 and P7?
oligo binding sites
53
what is DNA barcoding?
a technique that uses unique nucleic acid sequences (barcodes) to label and track individual cells or cell populations, enabling researchers to study their behavior, lineage, and interactions
54
what does DNA barcoding allow researchers to study about individual cells or cell populations?
- behavior
- lineage
- interactions
55
what is a technique that uses unique nucleic acid sequences to label and track individual cells or cell populations, enabling researchers to study their behavior, lineage, and interactions
DNA barcoding
56
what are some of the expanding roles of DNA Barcodes?
- evolution
- conservation
- ecology
- collections, data, and informatics
57
are there a higher amount of introns or exons from the nuclei? why?
introns because the transcripts have not been spliced yet
58
are there a higher amount of introns or exons from the nuclei? why?
exons because the introns have been spliced out during pre-mRNA processing
59
when doing an experiment, there is less sensitivity when beginning with a nuclei than a cell. Why?
there is a lower amount of RNA in the nuclei than the cell
60
there is a lower amount of RNA in the nuclei than the cell. How does this effect sensitivity?
it lowers sensitivity
61
nuclei use advantages:
- sample processing logistics
- less stress and mitochondrial signal
62
cell use advantages:
- more complete transcriptome
- detection sensitivity
- better connection with translation
63
the major steps in single-cell or nuclei data processing
1. count matrix
2. quality control
3. normalization
4. feature selection
5. integration
6. dimensionality reduction
64
what is a count matrix?
a table that summarizes the number of times specific RNA sequences (or reads) are found for each gene in a sample or set of samples
65
during data cleaning, what must be cleaned up?
doublets and ambient transcript signals
66
what are doublets?
a liquid droplet with two cells in it instead of one
67
what is ambient RNA?
RNA in the cell that it is not using / it is not associated with any particular cell population
68
why is ambient RNA an issue for single cell?
Ambient RNA can lead to misinterpretation of cell types, masked cell populations, and skewed gene expression patterns
69
is ambient RNA more problematic for nuclei or cell data?
nuclei because there will be more RNA around the nuclei and less around the cells
70
we use total cell counts to determine the quality of an individual cell. what is the issue with using a cell that has very few counts?
it is unlikely you will be able to identify the cell
71
what is the purpose of looking at mitochondrial counts?
- extreme amount of stress from the cell may not give the data you want
- less counts of the actual gene to identify function
72
define normalization
the practice of organizing data entries to ensure they appear similar across all fields and records
73
what is: the practice of organizing data entries to ensure they appear similar across all fields and records
normalization
74
why is normalization needed?
- sequencing depth
- RNA composition
- other technical bias
75
one of the reasons for normalization is because of RNA composition, what does that mean?
cells have a different number of a total amount of RNA because of the difference in cell size
76
what are the two types of batch effects?
- technical
- biological
77
what are batch effects?
- sequencing depth
- technologies
- sample quality
- technician
- cell cycle
78
what batch effect is biological?
cell cycle
79
what are principal components?
when a collection of points in a real coordinate space are a sequence of unit vectors
80
what is a principal components analysis?
a process of computing the principal components and using them to perform a change of bases on the data
81
what is a nearest neighbor graph?
a directed graph defined for a set of points in a metric space
82
define clustering
grouping a set of objects in such a way that objects in the same group are more similar to each other than to those in other groups
83
what is: grouping a set of objects in such a way that objects in the same group are more similar to each other than to those in other groups
clustering
84
define community
nodes refer to cells and cell-cell pairwise distances are applies in the Leiden algorithm (optimizing graph modularity locally on all nodes, then each small community is grouped into one node and the first step is repeated)
85
why is dimensionality reduction/feature selection needed?
with thousands of individual cells and genes per cell for each sample it is necessary to reduce the complexity of the data for visual inspection and to facilitate downstream clustering
86
PCA
principal components analysis projects a set of possibly correlated variables into a set of linear orthogonal variables
87
t-SNE
t-distributed stochastic neighbor embedding creates a probability distribution using the Gaussian distribution that defines the relationships between the points in high-dimensional space
88
UMAP
uniform manifold approximation and projection. a UMAP is contracted from a theoretical framework based in Riemannian geometry and algebraic topology.
89
what is the most common method for feature selection/dimensionality reduction?
UMAP
90
dimensionality reduction is highly __________
variable
91
what is the underlying concept of mapping cell clusters to cell identities?
a set of genes within a cluster of cells or nuclei will be significantly different in their level of expression compared to all other clusters of cells or nuclei
92
how do we identify cell types?
look at the state of the transcriptome and use prior literature to identify the cell type
93
define deconvolution
a process of resolving something into its constituent elements or removing complication in order to clarify it
94
what is: a process of resolving something into its constituent elements or removing complication in order to clarify it
deconvolution
95
what is the goal of deconvolution?
to estimate the proportion of a cell type present among a heterogenous mixture of cells using expressed marker genes that define a specific cell type
96
in genomics, what is deconvolution, in your own words?
estimating the percentage of cell types within a mixture of cells
