Single-Molecule Real-Time and Single-Cell RNA Sequencing

03.12.2018

Nina Beier, Maximilian Weingart

Outline

• Introduction• Genome, Transcriptome, Sequencing and Challenges

• Single-Molecule Real-Time Sequencing• Requirements, experimental setup and obtained data

• Single-Cell whole Transcriptome Sequencing• Transcriptome analysis, preparation from tissue to cDNA library

• Conclusion and Outlook

# 203.12.2018

Sequencing – What does it mean?

• Determination of primary structures of biopolymers

• Provides linear sequence of sub-structures

• Summarized much about atomic-level structure

• Especially interesting for DNA and RNA

# 303.12.2018

General structure of DNA

• Storage of genetic information of all living

organisms

• Built-up from monomeric units

nucleotides

• Certain sequences of nucleotides encode

genes

• Genes further encode e.g. proteins

# 403.12.2018

https://de.wikipedia.org/wiki/Desoxyribonukleins%C3%A4ure (02.12.2018)

Context of DNA and RNA

•Gene expression:

•Transcription: DNA RNA

•Translation: RNA Protein

• Genotype: Genetic composition of

an Organism

• Phenotype: Observable

appearance of an Organism

# 503.12.2018

https://www.technologynetworks.com/genomics/lists/what-are-the-key-differences-between-dna-and-rna-296719 (02.12.2018)

https://en.wikipedia.org/wiki/Gene_expression (02.12.2018)

Limitation of previous techniques

• Sanger method

• uses end tags and gel-electrophoresis for analysis

• Longer strands sequencing possible, but very long read times

• Alternative approaches (2nd generation sequencing):

• Faster overall throughput

• Sequence reads limited to ~400 nucleotides or shorter (e.g. Illumina)

• Often huge quantities are required for analysis

# 603.12.2018

Single-Molecule Real-Time Sequencing

# 703.12.2018

Single-Molecule Real-Time Sequencing

Idea: DNA polymerase as real-time sequencing machinefast, long reads

https://de.wikipedia.org/wiki/Datei:DNTP_nucleotide_incorporation_reaction.svg (02.12.2018)

03.12.2018 # 8

Single-Molecule Real-Time Sequencing

Phospholinked fluorescent nucleotides• Different colors to distinguish different nucleotides• Fluorescent dyes are released during incorporation of nucleotide

Pacific Biosciences Technology Backgrounder (11/24/2008)

natural DNA

03.12.2018 # 9

Single-Molecule Real-Time Sequencing

Journal of Applied Physics 103.3 (2008): 034301

nm

nm

Zero-Mode Waveguide (ZMW)• Sub-wavelength aperture• Observation volume confinement in zeptoliter (10−21) regime

Schematic of single ZMW Intensity distribution inside a ZMW

03.12.2018 # 10

Method

Science 323.5910 (2009): 133-138.

• DNA polymerase immobilized at bottom of ZMW, synthesizing DNA• Incorporation of fluorescenly labeled nucleotides produces light pulse• Record fluorescent signals over time

03.12.2018 # 11

DNA polymerasePhospholinked nucleotide

Detection

Pacific Biosciences Technology Backgrounder (11/24/2008)

03.12.2018 # 12

Measurements

Science 323.5910 (2009): 133-138.

Fluorescence time trace on linear template with 150 bases

03.12.2018 # 13

Properties of SMRT sequencing

• Long read lengths (possible up to > 30kbp)

• Short run times ( ~ 5 bases/s)

• No amplification needed

# 1403.12.2018

http://science.sciencemag.org/content/suppl/2008/11/20/1162986.DC1 (02.12.2018)

Real-time video of single molecule DNA polymerase activity measured on an array of 3,000 ZMWs.

10 μm

Single cell sequencingscRNA-seq method for whole transcriptome sequencing(based on Tang et al., 2009)

# 1503.12.2018

Steps of scRNA-seq

1) Isolation and lysis of single cells or single nuclei

2) Reverse transcription

3) cDNA amplification

4) Sequencing library preparation

5) Application of single-molecule sequencing

# 1603.12.2018

Isolation and capture of single cells

# 1703.12.2018

Hedlund, Deng (2018)

Reversed Transcriptase

• Synthesis cDNA from mature

mRNA

• Using poly-T primer with

anchor sequence (UP1)

• Digestion of unused Primers

# 1803.12.2018

Tang et al. (2009)

Reversed Transcriptase II

• Addition of poly-A tail to 3’-

end of first cDNA strand

• Synthesis of the second cDNA

strand

• Using again poly-T primers, but

different anchor sequence

(UP2)

# 1903.12.2018

Tang et al. (2009)

Amplification of cDNA

• Multipexing of cDNA using PCR

• Anchor sequences as primers

for DNA-Polymerase

# 2003.12.2018

Tang et al. (2009)

Applications

• Analysis of rare cell types and subpopulations

• Study of infectious diseases

• Characterisation of tumour cells

# 2103.12.2018

Summary and Outlook

Single Cell RNA-Sequencing (scRNA-seq):

• Direct Sequencing of RNA not possible

• Reversed Transcription from RNA to cDNA

• cDNA Amplification and Analysis Sequencing methods

• Increase throughput and accuracy

• Non-adenylation dependent methods

# 2203.12.2018

https://en.wikipedia.org/wiki/Single_cell_sequencing (02.12.2018)

Single-Molecule Real-Time Sequencing (SMRT):• Observing polymerase

during DNA synthesis

Possible due to:• Phospholinked fluorescent

nucleotides• Zero-Mode Waveguide

03.12.2018 # 23

Summary and Outlook

• Long read lengths • Short run times • No amplification needed

Applications:• Whole genome sequencing• De Novo sequencing• Monitor polymerase

dynamics• Single-Cell RNA sequencing• ....

03.12.2018 # 24

Summary and Outlook

Thanks for your Attention!

# 2503.12.2018

Bibliography

• Tang et al. (2009). mRNA-Seq whole-transcriptome analysis of a single cell. Nat. Methods 6, 377–382.• Kolodziejczyk et al. "The technology and biology of single-cell RNA sequencing." Molecular cell 58.4

(2015): 610-620.• Hedlund, Eva, and Qiaolin Deng. "Single-cell RNA sequencing: technical advancements and biological

applications." Molecular aspects of medicine 59 (2018): 36-46.• Wu, Angela R., et al. "Quantitative assessment of single-cell RNA-sequencing methods." Nature

methods 11.1 (2013): 41.• Eid, John, et al. "Real-time DNA sequencing from single polymerase molecules." Science 323.5910

(2009): 133-138.• Foquet, Mathieu, et al. "Improved fabrication of zero-mode waveguides for single-molecule

detection." Journal of Applied Physics 103.3 (2008): 034301.• Lundquist, Paul M., et al. "Parallel confocal detection of single molecules in real time." Optics letters

33.9 (2008): 1026-1028.• Roberts, Richard J., Mauricio O. Carneiro, and Michael C. Schatz. "The advantages of SMRT

sequencing." Genome biology 14.6 (2013): 405.• Pacific Biosciences Technology Backgrounder (11/24/2008)

https://www.ndsu.edu/pubweb/~mcclean/plsc411/Pacific%20Biosciences-technology_backgrounder.pdf

# 2603.12.2018