Single-cell analysis: basic principles

Jonathan Milhomens
Doutorando pelo Programa de Pós-graduação em Oncologia
Clínica, Células-tronco e Terapia Celular
Orientadora: Dra. Simone Kashima
 Outline

What is single-cell analysis?

Why study single-cells?
Single-cell RNAseq technology evolution
• Plate-based methods
• Microfluidics methods
Single-cell RNAseq data
 What is single-cell analysis?

Single cell studies and modeling approaches

have emerged in recent years, to understand
the biochemical processes in each individual
cell, as opposed to a large population of
cells and their average behavior. Habibi I, Cheong R, Lipniacki T,
Levchenko A, Emamian ES, Abdi A. Computation and measurement of cell decision making errors using single cell data.
PLoS Comput Biol. 2017;13(4):e1005436. Published 2017 Apr 5. doi:10.1371/journal.pcbi.1005436

Single cell analysis tools can be divided into

three groups: genomics, transcriptomics, and
proteomics. Hu P, Zhang W, Xin H and Deng G (2016) Single Cell Isolation and Analysis. Front. Cell
Dev. Biol. 4:116. doi: 10.3389/fcell.2016.00116
 Why study single-cells?

💡
Bulk Single-cell
analysis analysis
 Why study single-cells?

1. Small starting material

Allows the analysis from scarce and
precious samples.
(CTCs, tissue microdissection, PID)
 Why study single-cells?

2. Genome and transcriptome heterogeneity

Solves the genomic heterogeneity, gene
expression patterns and gene regulation in
individual cells

Grzywa, Tomasz M. & Paskal, Wiktor & Wlodarski, Pawel. (2017). Intratumor and Intertumor Heterogeneity in Melanoma. Translational Oncology. 10. 956-975. 10.1016/j.tranon.2017.09.007.
 Why study single-cells?
3. Statistical power
Comprises biological complexity of the samples, allowing accurate
measurements for each processed cell.

Velmeshev, D. et al. Single-cell genomics identifies cell type-specific molecular changes in autism. Science 364, 685-689, doi:10.1126/science.aav8130 (2019)
 Why study single-cells?
3. Statistical power
Bulk analysis can lead to false positives and miss important biological
divisions

Single-Cell Analysis - Powered by REPLI-g: Single Cell Analysis Series

Single-cell RNAseq technology evolution

Svensson, V., Vento-Tormo, R. & Teichmann, S. Exponential scaling of single-cell RNA-seq in the past decade. Nat Protoc 13, 599–604 (2018). https://doi.org/10.1038/nprot.2017.149
Single-cell RNAseq technology evolution
RNAseq plate-based methods

+ cDNA library processing

Picelli, S., Faridani, O., Björklund, Å. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171–181 (2014). https://doi.org/10.1038/nprot.2014.006
Single-cell RNAseq technology evolution

Svensson, V., Vento-Tormo, R. & Teichmann, S. Exponential scaling of single-cell RNA-seq in the past decade. Nat Protoc 13, 599–604 (2018). https://doi.org/10.1038/nprot.2017.149
Single-cell RNAseq technology evolution
DropSeq

Evan Z. Macosko, Anindita Basu, Rahul Satija, James Nemesh, Karthik Shekhar, Melissa Goldman, Itay Tirosh, Allison R. Bialas, Nolan Kamitaki, Emily M. Martersteck, John J. Trombetta, David A. Weitz, Joshua R.
Single-cell RNAseq technology evolution
DropSeq → Poisson distribution 😭

Evan Z. Macosko, Anindita Basu, Rahul Satija, James Nemesh, Karthik Shekhar, Melissa Goldman, Itay Tirosh, Allison R. Bialas, Nolan Kamitaki, Emily M. Martersteck, John J. Trombetta, David A. Weitz, Joshua R.
Single-cell RNAseq technology evolution
10X Genomics
Single-cell RNAseq technology evolution
RNAseq + protein quantification

Stoeckius M, Hafemeister C, Stephenson W, Houck-Loomis B, Chattopadhyay PK, Swerdlow H, Satija R, Smibert P. Simultaneous epitope and transcriptome measurement in single cells. Nat Methods. 2017
Single-cell RNAseq data
Data structure
Single-cell RNAseq data
Zero inflation!!!1!11!!!!
 Single-cell RNAseq data
In a two gene system, plotting the data is easy...

30

20

10

10 20 30
Stoeckius M, Hafemeister C, Stephenson W, Houck-Loomis B, Chattopadhyay PK, Swerdlow H, Satija R, Smibert P. Simultaneous epitope and transcriptome measurement in single cells. Nat Methods. 2017
 Single-cell RNAseq data
Dimensionality reduction

Zheng, G. X. Y. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 doi: 10.1038/ncomms14049 (2017)
 Single-cell RNAseq data
Deep biological information extraction

Zheng, G. X. Y. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 doi: 10.1038/ncomms14049 (2017)
 Single-cell analysis

Conclusions:
1. Applications are highly versatile
2. High dimensional data
3. Lower throughput and more expensive
than cytometry-based techniques
4. Gene expression spatial resolution***
Thank you
😊