THE NEXT GENERATION

SEQUENCING REVOLUTION
Genomics and its applications; single-cell
technologies

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 Sanger sequencing

https://www.abmgood.com/marketing/knowledge_base/next_generation_sequencing_introduction.php#sanger

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Sanger sequencing
• https://www.youtube.com/watch?v=jFCD8Q6qSTM&list=PL_Vc
B7OJ1TCAWRXN6vnC5lKbMHjlMtN8P&index=2

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 Our genome is 3 billion letters long!
(it takes 130 books to print out that many letters, even in font size 4!)

Images taken from Twitter

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Sequencing genomes (DNA) with NGS
technology is now easy, fast, and cheap

1990 – 2003
13 years, $1 billion USD Today
1 day, $100 USD

Images taken from NIH Human Genome Research Institute

and Illumina

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Sequencing genomes (DNA) with NGS
technology is now easy, fast, and cheap

1990 – 2003
13 years, $1 billion USD Today
1 day, $100 USD

Images taken from NIH Human Genome Research Institute

and Illumina

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 Human Genome Project
• Used Sanger Sequencing
• Took ~13 years (started in 1985!!!)
• Spent ~3 billion USD!!!
• Today: ~100 USD

DNA sequencing costs: data from the NHGRI Genome Sequencing

Program (GSP). http://www.genome.gov/sequencingcosts/.

Nature editorial staff (2010). Human genome at ten: The sequence

explosion. Nature, 464, 670-671. doi:10.1038/464670a

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Next-generation sequencing (NGS)
• Massively parallel
• Huge output of data
• Decreasing costs
• Fast
• https://www.youtube.com/watch?v=jFCD8Q6qSTM&list=PL_Vc
B7OJ1TCAWRXN6vnC5lKbMHjlMtN8P&index=2

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 Roche 454: Pyrosequencing

Jacopo Pompilii, DensityDesign Research Labhttps://commons.wikimedia.org/w/index.php?curid=37083509

http://bitesizebio.com/19008/how-bisulfite-pyrosequencing-works/

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Roche 454: Pyrosequencing
https://youtu.be/jFCD8Q6qSTM?t=3m40s`

ML Metzker, Nature Review Genetics, 2009

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 https://youtu.be/jFCD8Q6qSTM?t=6m42s
Ion Torrent

http://www.genomics.cn/en/navigation/show_navigation?nid=2640

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 Illumina: Sequencing-by-synthesis (SBS)

http://www.3402bioinformaticsgroup.com/service/

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Illumina: Sequencing-by-synthesis (SBS)
• To review the details and video:
https://www.illumina.com/science/technology/next-generation-
sequencing/sequencing-technology.html

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 BGI (华大基因)/CompleteGenomics DNA
nanoball technology

All the images taken from mgi-tech.com

1. Make the DNA 2. Load the

nanoball nanoball array

A helpful video: https://www.bilibili.com/video/BV1Ub4y1S7Z6/

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BGI (华大基因)/CompleteGenomics DNA
nanoball technology

3. Read out the DNA nanoball sequence All the images taken from mgi-tech.com

A helpful video: https://www.bilibili.com/video/BV1Ub4y1S7Z6/

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 Pacific Biosciences: SMS

ML Metzker, Nature Review Genetics, 2009

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Pacific Biosciences: SMS
• https://www.youtube.com/watch?v=NHCJ8PtYCFc&list=PL_Vc
B7OJ1TCAWRXN6vnC5lKbMHjlMtN8P&index=4

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 4th Generation Sequencing!!!
• NANOPORES!!!

Schaffer, MIT Technology Review, 2012

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Nanopore Sequencing
• Rated lifetime of one MinION flow cell: 48 hours of run time

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Oxford Nanopore
• https://www.youtube.com/watch?v=CE4dW64x3Ts&index=5&li
st=PL_VcB7OJ1TCAWRXN6vnC5lKbMHjlMtN8P

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 Which sequencing
Project platform(s) would you Why?
propose?
De novo sequencing and
assembly of a microbial
genome

Sequencing a plasmid

Re-sequencing a human
genome (e.g. cancer sample) to
look for novel mutations

Rapid diagnosis of a viral

infection by sequencing in the
field

Targeted amplicon/exome
sequencing

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Applications of NGS
• Your ideas
• Anyone working on NGS projects right now?

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Applications of NGS
• Cancer research
• Pre-natal diagnostics
• Discovery of new microbial or viral species
• Predicting organ transplant rejection

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Applications of NGS
• Cancer research and diagnosis
• Personal cancer genomes
• RNA-seq comparing normal tissue to cancer tissue

• E.g. Breast Cancer types

• ER+ or PR+ (drug tamoxifen to block hormone receptors)
• HER2+ (drug herceptin)
• Triple positive
• Triple negative (often BRCA1+; chemo, high chance of relapse)

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 Applications of NGS
• Pre-natal diagnostics
• DNA
• RNA

From Ariosa website

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 Applications of NGS
• Pre-natal diagnostics
• DNA
• RNA

Lillian M. Zwemer, and Diana W. Bianchi Cold Spring Harb Perspect Med 2015;5:a023101

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Applications of NGS
• Predicting organ transplant rejection
• DNA of donor
• RNA of microbes and viruses

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 Applications of NGS
• Predicting organ transplant rejection
• DNA of donor
• RNA of microbes and viruses

De Vlaminck et al., Science Translational Medicine, 2014

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Applications of NGS
• Discovery of new microbial or viral species
• De novo assembly

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Applications of NGS
• Tools in the research lab: WGS, WES, RNA-seq, ChIP-seq,
CHIRP-seq, methyl-seq, Hi-C, PRO-seq, ATAC-seq… etc.

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 RNA-seq

Image from BiteSize Bio

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 RNA-seq
Example of data

Which of the three transcripts is expressed with highest abundance?

Schulz MH, et al., Bioinformatics, 2012

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 RNA-seq
Example of data

Schulz MH, et al., Bioinformatics, 2012

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EPIGENOMICS USING NGS

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ChIP-seq

• ChIP: assesses protein-DNA

Fix & lyse cells
interactions

Fragment
chromatin

IP & wash
Input: no
IP

Extract & analyze

DNA

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 ATAC-seq

Interrogates
chromatin
accessibility

Easy to perform
(compared to
FAIRE-seq,
MNase-seq,
DNAse-seq etc.)

Buenrostro J.D., et al., Nature Methods, 2013

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 Hi-C Probes 3D conformation of the genome architecture

Rao et al., Cell, 2014

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SINGLE-CELL OMICS
How does NGS at the SINGLE-CELL level lead to
new biological and medical discoveries?

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Why single cell?
• Tissues consist of heterogeneous cell types
• Method can be used for rare/valuable cell types
• e.g. circulating tumor cells; primary embryonic tissues

Heterogeneous cell
population

ß stem cell!

Population-level average
Single-cell profiling
from bulk profiling

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Single-cell genomics workflow

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SINGLE CELL RNA-SEQ

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 Many technology platforms to choose from
beads with barcode

t Han
ghpu ds
-o
ou

n
Dissociation

Th

Tim
e
Mouth Pipet Droplets & Microwell

Microfluidics FACS sorting/Robot

Wu, Wang, Streets, and Huang, Annual

Review of Analytical Chemistry, 2017

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Captured cells in the C1

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LIFS 6170
 Single cell transcriptomics methodology
Poly(A)-tailing
AAAAAAA
+TdT TTTT + TTT TTTT
4

2nd Strand cDNA Synthesis

TTT
AAA AAA

Template switch PCR

+MMLV/SS2 +Pol
1 3a + GGG
AAAAAAA
TTTT 4 +
XXX

XXX
GGG
Dissociation CCC XXX

Paritial Digestion
+RNase TTTT +Pol TTTT
+ Ligase

2 IVT+PCR
Lysis & RT +
4 +T7 pol + N6 +
AAAAAAA N6
TTTT
RT PCR
Direct Amplification

WGA
RNA Anchor / T7 promoter
+Phi29 pol N6
DNA T7 polymerase
N6
3b + N6 N6
Cell strand synthesis
N6
Amplification
Upper sequence is 5’->3’
Lower sequence is 3’->5’
Wu, Wang, Streets, and Huang, Annual
Review of Analytical Chemistry, 2017

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What does the data look like?

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 What does the data look like?

Camara P. G.., 2018, Current Opinions in Systems Biology

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The Human Cell Atlas
A “Google Maps” For the cells in the human body

…Can it really be done? How?

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 Many technology platforms to choose from
beads with barcode

t Han
ghpu ds
-o
ou

n
Dissociation

Th

Tim
e
Mouth Pipet Droplets & Microwell

Microfluidics FACS sorting/Robot

Wu, Wang, Streets, and Huang, Annual

Review of Analytical Chemistry, 2017

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 Microfluidic droplets applied to NGS
• Using droplets as chambers, we can increase throughput even
more, to ~100,000 single cells per run!
• Two Harvard groups published similar technology recently:
• Drop-seq - https://vimeo.com/128484564
• inDrop - https://vimeo.com/126829858

Drop-seq: http://www.sciencedirect.com/science/article/pii/S0092867415005498
inDrop: http://www.cell.com/cell/fulltext/S0092-8674(15)00500-0

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Microfluidic droplets applied to NGS

“Barnyard
experiment”

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Single-cell resolution profiling of a whole organism!

RESEARCH ARTICLE

Cite as: N. Karaiskos et al., Science

10.1126/science.aan3235 (2017).

The Drosophila embryo at single-cell transcriptome

resolution
Nikos Karaiskos,1* Philipp Wahle,2* Jonathan Alles,1 Anastasiya Boltengagen,1 Salah Ayoub,1 Claudia Kipar,2
Christine Kocks,1 Nikolaus Rajewsky,1† Robert P. Zinzen2†
1
Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz
Association (MDC), 13125 Berlin, Germany. 2Systems Biology of Neural Tissue Differentiation, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center
for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany.
*These authors contributed equally to this work.
†
Corresponding author. Email: [email protected] (N.R.); [email protected] (R.P.Z.)

By the onset of morphogenesis, Drosophila embryos consist of about 6000 cells that express distinct

Downloaded from http://science.sciencemag.org/ on November 1

gene combinations. Here, we used single-cell sequencing of precisely staged embryos and devised
DistMap, a computational mapping strategy to reconstruct the embryo and to predict spatial gene
expression approaching single-cell resolution. We produce a virtual embryo with about 8000 expressed
genes per cell. Our interactive “Drosophila-Virtual-Expression-eXplorer” (DVEX) database generates
three-dimensional virtual in situ hybridizations and computes gene expression gradients. We used DVEX
to uncover patterned expression of transcription factors and long noncoding RNAs, as well as signaling
pathway components. Spatial regulation of Hippo signaling during early embryogenesis suggests a
mechanism for establishing asynchronous cell proliferation. Our approach is suitable to generate
transcriptomic blueprints for other complex tissues.

Intricate gene regulatory networks produce and maintain tial gene expression patterns emerge as cells translate an-
complex assemblies of specialized cells such as tissues and teroposterior and dorsoventral positional information into
organs. To unravel the underlying gene expression dynam- transcriptional responses [e.g., (22)]. Stage 6 is marked by
ics, significant efforts have been made to compare tissue- the first morphogenetic movements after cellularization
specific materials (1–4). Cell culture often constitutes a poor completes and gene expression around this stage has been
proxy for in vivo complexity, and dissected tissues are com- extensively assayed in whole embryos (e.g., (23), in mutants
prised of heterogeneous cell populations (3–10). An alterna- converting entire embryos to germ layers (4) and in dissect-
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Fig.ed slices
tive is isolation of specific cell types via cell sorting (11–14); 1. (24).
De- and
Available in situ reconstructing
databases present systemati-the embryo by single-cell
Single cell resolution profiling of a whole organism!
Fig. 5. Prediction accuracy and
detection of new regulators. (A)
vISH predictions are accurate
across a wide variety of expression
patterns. Expression of CGs had not
been reported previously. (B)
Patterned expression of putative
transcription factors. (C) Patterned
expression of lncRNAs. (D) CR43432
and pan-neurogenic genes are
expressed in complimentary

Downloaded from http://science.sciencemag.org/ on No

patterns. Dual vISH of SoxN and
CR43432 (top left), double in situ
hybridization validates the
predicted expression. CR43432 is
additionally expressed in yolk
nuclei (not shown in vISH).

Fig. 5. Prediction accuracy and detection of new regulators. (A) vISH

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 Single-cell analysis of 20 mouse tissues –
mouse cell atlas

Tabula Muris Consortium. (2018). Single-cell transcriptomics of 20 mouse

organs creates a Tabula Muris. Nature, 562(7727), 367.

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 Single-cell analysis of 20 mouse tissues –
mouse cell atlas

Tabula Muris Consortium. (2018). Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature, 562(7727), 367.

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Single-cell analysis of 2 million cells from
developing mouse embryo

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Aging mouse atlas

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 Dr. Qiuyu Jing Dr. MING Jingsi Prof. Can YANG Prof. Zhixiang LIN
HKUST LIFS East China Normal U. HKUST MATH CUHK

TABULA MICROCEBUS:
A SINGLE CELL ATLAS OF THE
GREY MOUSE LEMUR
Work from our lab:
Two LCA Manuscripts under review at Natur
Zhao et al., Nat. Comp. Sci. 2022
Ming et al., Brief Bioinform., 2022
Olivieri et al., eLife, 2021

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Mouse Lemur Single Cell Atlas (Tabula
Microcebus)

The first non-human primate

cell atlas to be released

tabula-microcebus.ds.czbiohub.org

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Mouse Lemur Single Cell Atlas (Tabula
Microcebus)

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Mouse Lemur Single Cell Atlas (Tabula
Microcebus)

226,701 total pass QC cells

~95% from 10X, 5% from SS2

27 tissues/organs

256 cell names

All-tissue UMAP (left) generated

using FIRM

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Adipose tissue:A unique metabolic ‘organ’
• Mouse lemur are the only
primates that ‘hibernate’ （冬眠）
• Termed ‘torpor’
• Increased body weight, decreased
metabolism
• Conventionally, mouse and
human have two main types of
adipose tissue (ADIPOQ+):
• White adipose tissue （白色脂肪）
- expresses ADIPOQ, APOE,
NNAT
• Brown adipose tissue （棕色脂肪）
- expresses UCP1, CIDEA, CFD
• (Beige fat 米色脂肪)

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Gene expression of fat cells in lemur is unique:
distinction between WAT and BAT is blurred

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Spermatogenesis: naturally a continuous
trajectory
Mammalian spermatogenesis is a continuous and irreversible differentiation
process from spermatogonial stem cells (SSCs) to sperm cells

Figure from Hermann et al., Cell Reports, 2018

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A gradient of cells in the lemur testis
diplotene spermatocyte

round spermatid

pachytene spermatocyte
10
spermatogonium
early spermatocyte
UMAP_2

pachytene spermatocyte
diplotene spermatocyte
round spermatid
elongating spermatid
elongating spermatid
elongated spermatid
0

elongated spermatid
early spermatocyte

spermatogonium

−5 0 5 10 15
UMAP_1

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Papers to read:
• Review papers on single-cell RNA-seq;
• Best practices in scRNA-seq data analysis
• Please try out the tutorial from Sanger:
https://www.singlecellcourse.org/index.html

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SINGLE CELL WHOLE
GENOME SEQUENCE (WGS)

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 Microfluidic droplets applied to NGS
• Single cell DNA sequencing using Multiple Displacement Amplification (MDA) is
known to have problems of amplification bias (e.g. preference for GC rich regions)
• Huang group at Peking University solves this problem using droplet-based MDA
(http://www.pnas.org/content/112/38/11923.full)

Fu et al, PNAS, 2015

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Microfluidic droplets applied to NGS

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 Single-cell proteomics CYTOF – cytometry and time-of-flight

Image: http://cytof.scilifelab.se/homepage/static/images/cytof.jpg
Review: Bendall, Sean C., and Garry P. Nolan. "From single cells to deep phenotypes in cancer." Nature biotechnology 30.7 (2012): 639-647.

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 Single-cell multi-omics
Ab-seq or CITE-seq: cellular indexing of transcriptomes and epitopes by sequencing

Stoeckius, Marlon, et al. "Simultaneous epitope and transcriptome

measurement in single cells." Nature 201 (2017): 7.

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 Single-cell multi-omics
scTrio-seq

Hou, Yu, et al. "Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic
heterogeneity in hepatocellular carcinomas." Cell research 26.3 (2016): 304-319.

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