004 (FRA) 2021 ScRNAseq Methods Data Analysis For Bioinformatics

scRNA-seq
methods and
data analysis
A brief introduction… by
PhD, Silvia Giulia Galfrè
History
Molecular Biology and Genomics – a.a. 2021-2022

• Taeg et al. 2009
• Early developmental stage

embryo: just few cells
• Number of publications last

year: around 30.000
• Different methods: more than

50…
From (Svensson, et al., 2018) with some integration and modification, Plot of some important scRNA-
seq experiment reporting the analyzed cell number and the year.
3
La Sapienza University
Cell atlases

• Now there is a great effort to construct
different comprehensive single cell atlases
• Examples:
• Human cell atlas
https://www.humancellatlas.org/
• Mouse Tabula Muris
https://tabula-muris-senis.ds.czbiohub.org/
• Mouse cell atlas: >520,000 single cells from
>10 Mouse tissues -
http://bis.zju.edu.cn/MCA/index.html
• Drosophila Atlas: https://flycellatlas.org/
• … many more for tissues
4
From
Why single cell?

Bulk RNA-seq scRNA-seq
• Precise single cell
• Average
information
information
• Identification of
• Information for
rare cell types
rare cell types
lost • More difficult
• Easier to obtain • More expensive
• Cheaper • Many methods
• Quite standard • No standard
workflow workflow
5
General workflow
1. Cell isolation 2. Library preparation
3. Data analysis
There are many methods...

• They differ for cell isolation
protocols
• Reaction “chamber”
• And library preparation
protocols
• Notice that FACS-sorting can be
used before other protocols like
Fluidigm or micro-fluidic
protocols, or alone to isolate and
sort cells.
7
In wells methods
Advantage: ease the control of the reactions
Drawback: small amount of cells
8
Fluidigm C1

Complete automated commercial technology
• analyze only 96
cells each round 1
• full-length RNA
sequences
• SMARTer® First, the primer poly-T hybridizes with the mRNA poly-A tail and the reverse
chemistry for cDNA transcriptase can produce the cDNA.
synthesis (Gong, et
al., 2018)
9
Fluidigm C1

• analyze only 96
cells each round 1
• full-length RNA
sequences
2
• SMARTer®
chemistry for cDNA
synthesis (Gong, et When the RNA end is reached, the enzyme’s terminal transferase activity adds some
al., 2018) deoxycytidines. These can base pair with the poly-G of another primer present in
solution that can be used for a second round of polymerisation.
10
Fluidigm C1

• analyze only 96
cells each round 1
• full-length RNA
sequences
2
• SMARTer®
chemistry for cDNA
synthesis (Gong, et 3
al., 2018)
The yellow primers are used for the subsequent PCR amplification.
11
Fluidigm C1

Tagmentation step
This is necessary to break
1
the cDNA keeping the
positional information of
the different pieces. This
is done through a 2
genetically modified Tn5
enzyme that can cut a 3
double stranded DNA
inserting at both sides a
specific adapter.
Then all cDNA is pooled 4

and sequenced.
12
SMARTer total RNA

Fluidigm C1 instruments
• derived from the previous

• library preparation after removing the rRNA
(or not)
• full-length sequencing is recovered by using
random primers for template switching
• polyadenylated and non-polyadenylated
RNA molecules
• preserve strand-of-origin information
13
La Sapienza University From https://www.takarabio.com/
Smart-seq3

• Poly-A+ method
• Full length
• This method allows to do a direct allelic
RNA counting trough SNPs detection
• UMI inserted in a set of barcodes added at
the 5’-end
From Hagemann-Jensen, et al., 2020
14
UMI

RNA
molecules
Unique Molecular Identifier
• First introduced in 2014

cDNA • They are short sequences used to uniquely
formation tag each molecule in a sample library
• Fundamental to count each molecule once
UMI
without bias from PCR
After PCR
1 molecule each gene

Deduplication
2 molecules
Islam, S., Zeisel, A., Joost, S. et al. Quantitative single-cell RNA-seq
with unique molecular identifiers. Nat Methods 11, 163–166 (2014).
15
SPLiT-seq

Split-pool barcoding
• a suspension of formaldehyde fixed cells or

nuclei through four rounds of combinatorial
barcoding
• third-round barcode -> also UMI
• Four rounds of combinatorial barcoding can

yield 21,233,664 barcode combinations (three
rounds of barcoding in 96-well plates followed
by a fourth round with 24 PCR reactions)
16
Rosenberg, Alexander B., et al Science 360.6385 - 2018
SPLiT-seq

• 156.049 single-nucleus transcriptomes
• P2 and P11 mouse brains and spinal cords
• Around100 cell types were identified
17
La Sapienza University Rosenberg, Alexander B., et al Science 360.6385 - 2018
Insert or Drag and Drop your Image
Droplet based
Advantages: high number of cells, not
expensive
Drawback: not full length, poly A
18
General features

• All high-throughput: from hundreds to thousands of cells
• Bead plays a fundamental role: primers are 5’-end linked to the bead with a cleavable linker
• Each primer contains a PCR primer, a cell barcode (the same for all the primers on the bead
but different between different beads), a random UMI and a poly-T sequence
• Water-oil emulsion: each aqueous drop will contains a bead and a single cell (or nothing, or
just a bead).
Zhang, Xiannian, et al. "Comparative analysis of droplet-based ultra-high-throughput single- 19

cell RNA-seq systems." Molecular cell 73.1 (2019): 130-142.
General steps

• Inside the drop, the cell lyses and the mRNA poly-A tails
hybridize with the poly-T tail of the primers attached to the
bead.
Macosko, et al., 2015

20
General steps

hybridize with the poly-T tail of the primers attached to the
bead.
• Then, reverse transcription occurs, and all the cDNAs are
pooled and sequenced.

21
General steps

hybridize with the poly-T tail of the primers attached to the bead.
• Then, reverse transcription occurs, and all the cDNAs are pooled
and sequenced.
• All sequences coming from different cells are mixed together, but
thanks to the cell barcode inserted, it is possible to demultiplex
the data, separating the sequences coming from different cells.

Zhang, Xiannian, et al. "Comparative analysis of droplet-based ultra-
22
high-throughput single-cell RNA-seq systems." Molecular cell 73.1
La Sapienza University (2019): 130-142.
In Drop

Klein, et al., 2015 – citations 2229
Barcoded
• Beads: hydrogel Primer
Bead
• Link between primers and bead: UV
cleavable
• Reverse transcription: directly inside each
drop
• cDNA amplification (it uses the CEL-seq Droplet
protocol with a linear in vitro transcription) Generation
quite time-consuming.
Reaction in
Droplets 2.5h
Reaction after 28h 23

Demulsification
Drop-seq

Macosko, et al., 2015 – citation 4303
Barcoded
Primer
• Resin beads Bead
• mRNA capture efficiency is quite low
because primers are attached only to the
surface of the bead
• mRNA is captured inside the drop, reverse
transcription > later Droplet
Generation
• cDNA amplification it uses a direct
template-switching protocol
Reaction in
Droplets 0.3h
Reaction after
Demulsification 9h 24
10X Genomics (chromium)

Zheng, et al., 2017 – citations 2454
Barcoded
Primer
• Hydrogel beads -> they can be dissolved
Bead
• Reverse transcription directly inside each
drop
• For cDNA amplification it uses a direct
template-switching protocol
• commercial Droplet
Generation
Reaction in
Droplets 1h
Reaction after
Demulsification 7h 25
Methods comparative chart

Method Type Cell number RNA full lenght UMI RNA species
Fluidigm C1 in wells low throughput yes no poly-A
SMARTer total
RNA in wells low throughput yes no total
Smart-seq3 in wells low throughput yes yes poly-A
SPLiT-seq in wells high throughput no yes poly-A
InDrop dropplets high throughput no yes poly-A
DropSeq dropplets high throughput no yes poly-A
10X dropplets high throughput no yes poly-A
26
Workflow: part 1-Data Pre-processing

READS QUALITY CONTROL
1. the number of counts

per barcode (count
depth)
2. the number of genes
per barcode
3. the fraction of counts
from mitochondrial
genes per barcode
Seurat official web-site 27

Workflow: part 1-Data Pre-processing

READS QUALITY
CONTROL
1. the number of counts per

barcode (count depth),
2. the number of genes per
barcode,
3. the fraction of counts from
mitochondrial genes per
barcode
Large image
Practical part 1A
- file: 10X_Seurat.Rmd
Matrix examples

Drop-seq 10x
Very sparce matrixes

30
Methods features

• Technical noise: variation given by experimental
randomness
10x Drop-seq inDrop
• Precision: system resolution - nearest correlation • Sensitivity: transcripts detection also for
between one cell the others low level of expression
Zhang, X. et al. Comparative Analysis of Droplet-Based Ultra-High-Throughput Single-Cell RNA-Seq Systems. Mol. Cell 73, 130-142.e5 (2019).
Workflow: part 1. Data normalization

1. Most common : “count per million” CPM . It
assume that all cells initially contain the same
amount of RNA
2. Same assumption: down sampling protocol.
3. Scran’s pooling-based size factor estimation
method: size factors are estimated based on a
linear regression over genes, after cells are
pooled to avoid technical dropout effects
4. Non-linear normalization methods (for more
complex unwanted variation). Many such
methods involve the parametric modelling of
count data (negative binomial or Poisson-
gamma).
We cannot expect that a single normalization method is
appropriate for all types of scRNA-seq data.
Computational Methods for Single-Cell RNA Sequencing
Brian Hie, Joshua Peters, Sarah K. Nyquist, Alex K. Shalek, Bonnie Berger, Bryan D. Bryson
Annual Review of Biomedical Data Science 2020 3:1, 339-364
Log transformation

Three effects:
1. distances between log-transformed expression values represent fold changes
2. mitigating the mean–variance relationship in single-cell data
3. reducing the skewness of the data (approximation to normally distributed)
log transformation of normalized data can introduce spurious differential expression

effects into the data
Gene scaling ??

Seurat tutorial --- Yes
Others no
Should all genes be weighted equally for downstream analysis, or the magnitude of
expression of a gene is an important information?
Large image
Practical part 1B
- file: in_wells_Seurat.Rmd
Dropplet batch effect
Molecular Cell 2019 73130-142.e5DOI: (10.1016/j.molcel.2018.10.020)

Different methods for dataset
integration

Large image
Practical part 2B
- file: in_wells_Seurat.Rmd
Workflow: part 3

Dimensionality reduction
1. To capture the underlying structure in

the data in as few dimensions as
possible
2. Identifying highly variable genes is
primarily used to focus downstream
dimensionality reduction and
clustering
41
Workflow: part 3 PCA –

Principal Component Anaysis

possible
2. PCA is a linear decomposition
dimensionality reduction and clustering
42
Workflow: part 3 PCA –

Principal Component Anaysis

possible
44
Workflow: part 3 t-SNE -

t-distributed stochastic neighbor
embedding

possible
4. Non-linear: richer structural information
to prevent dense overcrowding within a
visualization but may also introduce
unrepresentative distortion
45
Workflow: part 3 UMAP -

Uniform Manifold Approximation
and Projection

possible
4. Non-linear: richer structural
information to prevent dense
overcrowding within a visualization but
may also introduce unrepresentative
distortion
46
Workflow: part 3

Cell clustering, identification and DE
• Grouping cells based on similarity of gene

expression profiles
• Clustering techniques can be split into
several general categories
• Fundamental before cell identification
• Different clustering methods lead to
different cell identification (especially
important if we want rare cells)
• Cluster assignment with marker genes
• After cluster identification => differential
expression
47
Large image
Practical part 3A
- file: 10X_Seurat.Rmd
Workflow: part 4

dataset exploration
49
Large image
Practical part 4A
- file: 10x_Seurat.Rmd
Cellular trajectories

Pseudotime approches
• It aim to arrange cells along
an axis of variation that
depends on both cells and
genes
• often applied to cells
undergoing differentiation
• cell orderings can be
meaningful in many
experiments (as spatial
gradients or response to
external stimuli)
• Many different algorithms
Saelens, W., Cannoodt, R., Todorov, H. et al. A comparison of single-cell trajectory inference methods. Nat
Biotechnol 37, 547–554 (2019). https://doi.org/10.1038/s41587-019-0071-9
La Sapienza University 53
Cellular trajectories

One of the most easy and
common: Monocle
54
Thank Silvia Giulia Galfre
You silvia.galfre@uniroma1.it

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scRNA-seq

Molecular Biology and Genomics – a.a. 2021-2022

• Early developmental stage

• Number of publications last

• Different methods: more than

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

Then all cDNA is pooled 4

Molecular Biology and Genomics – a.a. 2021-2022

• derived from the previous

Molecular Biology and Genomics – a.a. 2021-2022

From Hagemann-Jensen, et al., 2020

Molecular Biology and Genomics – a.a. 2021-2022

• First introduced in 2014

1 molecule each gene

Molecular Biology and Genomics – a.a. 2021-2022

• a suspension of formaldehyde fixed cells or

• third-round barcode -> also UMI

• Four rounds of combinatorial barcoding can

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

Zhang, Xiannian, et al. "Comparative analysis of droplet-based ultra-high-throughput single- 19

Molecular Biology and Genomics – a.a. 2021-2022

Macosko, et al., 2015

Molecular Biology and Genomics – a.a. 2021-2022

Macosko, et al., 2015

Molecular Biology and Genomics – a.a. 2021-2022

Macosko, et al., 2015

Molecular Biology and Genomics – a.a. 2021-2022

Reaction after 28h 23

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

1. the number of counts

Seurat official web-site 27

Molecular Biology and Genomics – a.a. 2021-2022

1. the number of counts per

Molecular Biology and Genomics – a.a. 2021-2022

Very sparce matrixes

Molecular Biology and Genomics – a.a. 2021-2022

10x Drop-seq inDrop

Molecular Biology and Genomics – a.a. 2021-2022

Molecular Biology and Genomics – a.a. 2021-2022

log transformation of normalized data can introduce spurious differential expression

Molecular Biology and Genomics – a.a. 2021-2022

Insert or Drag and Drop your Image

Molecular Cell 2019 73130-142.e5DOI: (10.1016/j.molcel.2018.10.020)

Insert or Drag and Drop your Image

Molecular Biology and Genomics – a.a. 2021-2022

1. To capture the underlying structure in

Molecular Biology and Genomics – a.a. 2021-2022

1. To capture the underlying structure in

Molecular Biology and Genomics – a.a. 2021-2022

1. To capture the underlying structure in

Molecular Biology and Genomics – a.a. 2021-2022

1. To capture the underlying structure in

Molecular Biology and Genomics – a.a. 2021-2022