DNA Sequencing Costs and Technologies

A/Prof Jeffrey Funk
Division of Engineering
and Technology Management
National University of Singapore
For information on other technologies, see http://www.slideshare.net/Funk98/presentations

 Identify the sequence
and identity of 3 billion
base pair nucleotides in
DNA strand
 Nucleotides encode the
genetic instructions for
organisms
 Four types of nucleotides
in a DNA strand
◦ Adenine
◦ Thymine
◦ Cytosine
◦ Guanine

 Can the Falling Cost of sequencing and
synthesizing DNA
◦ How can we use the data?
 Enable us to reduce the cost and time of
developing better
◦ Drugs? Is personalized medicine possible?
(medicine has recognized about 6,000 diseases
that can be traced to one or more genes)
◦ Crops? Can we feed the world?
◦ Bio-fuels? Can we reduce carbon emissions?
◦ Bio-Materials? Are better materials possible?
◦ Complex biological systems? Can we create computers
from biological parts?
http://www.economist.com/news/briefing/21661799-it-now-easy-edit-genomes-plants-animals-and-humans-age-red-pen

Session Technology
1 Objectives and overview of course
2 How/when do new technologies become economically feasible?
3 Two types of improvements: 1) Creating materials that better
exploit physical phenomena; 2) Geometrical scaling
4 Semiconductors, ICs, electronic systems
5 Sensors, MEMS and the Internet of Things
6 Bio-electronics, Health Care, DNA Sequencers
7 Lighting, Lasers, and Displays
8 Roll-to Roll Printing, Human-Computer Interfaces
9 Information Technology and Land Transportation
10 Nano-technology and Superconductivity
This is Sixth Session of MT5009

 Why do Costs Fall and Other Improvements
Occur?
 New Methods Continue to Emerge
◦ Pyrosequencing (454 Life Sciences/Roche and
Illumina)
◦ Single-molecule real-time sequencing (Pacific Bio)
◦ Semiconductor arrays (Ion Torrent)
◦ Nanopores (Oxford Nanopore Technologies)
◦ Methods of data compression
 Synthesizing DNA
 Who Cares? What are the Implications?
 Conclusions

 New methods of sequencing
◦ Maxam-Gilbert Sequencing: relies on cleaving of
nucleotides by chemical methods
◦ Chain Termination methods (sometimes called
Sanger method): bases are illuminated with UV light,
read with X-rays
◦ Dye-termination: reading sequences with fluorescent
dyes where each nucleotide emits light in different
wavelengths
 Improved lasers and cameras to read
fluorescent dyes
 More parallel processing
 Smaller feature sizes, reductions in scale
http://www.dnasequencing.org/history-of-d

Source: High Throughput Sequencing Technologies, Brian Krueger,
http://www.slideshare.net/Kruegsybear/high-throughput-sequencing-technologies-on-the-path-to-the-0-genome
Dye-
Termination

Source: Nature Biotechnology 30(11), 1023-1026, November 2012
Many new approaches are being investigated

 This can be understood by reading highly cited
papers such as
◦ “Genome sequencing in micro-fabricated high-
density pico-liter reactors” (Margulies, 2005) and
◦ “Toward nano-scale genome sequencing” (Ryan et al,
2007)
 Quote from Ryan et al: “The ability to construct nano-
scale structures and perform measurements using novel
nano-scale effects has provided new opportunities to
identify nucleotides directly using physical, and not
chemical, methods.”
 In fact, just the titles of these papers are fairly
suggestive. In all of these decreasing scale
examples, totally new forms of equipment,
processes and factories were required.

 Read lengths
 Accuracies
 Speeds
 Improvements in these variables also lead
to reductions in cost of sequencing
 Capability to analyze and use gathered
data
◦ need better computers
◦ need more storage

Improvements in Output per Instrument Run
Nature 2011, 470: 198-203, Elaine Mardis

 http://www.youtube.com/watch?v=6ldtdWjD
wes&list=PL7DFC2F1730BD5F3A
 http://www.youtube.com/watch?v=iz2xh8U9j
Yc&index=3&list=PL7DFC2F1730BD5F3A

 1) separate DNA into smaller strands
 2) make copies of strands (i.e., amplification) with
emulsion beads in plastic containers (need redundancy
for higher accuracy)
◦ done with small containers on large wash plate so that many
copies are made in parallel
◦ smaller containers and larger wash plates lead to more parallel
and faster processing
 3) identify DNA nucleotides utilizing lasers and cameras
◦ Nucleotides (A, T, C G) emit light in presence of an enzyme,
ADT (Adenosine Triphosphate)
◦ falling costs of lasers and cameras reduce costs
 4) Analyze data with computers

One source: http://www.454.com/downloads/news-events/how-genome-sequencing-is-done

 Make copies to improve accuracy through
redundancy
 454 PicoTiterPlate from LifeSciences
◦ contains 1.6 million hexagonal wells
◦ each holds 75 pico-liters (10-12 liters, <100 micron diameter)
 These wells can be made much smaller
◦ dimensions on integrated circuits (ICs) are on the order of
20 nano-meters
◦ Is it possible to reduce feature sizes by 1000 times or
volumes by 109

Fluorescent Dyes,
Lasers, and Cameras
(Step 3)
As bases move across wash
plate during sequencing run, a
nucleotide (molecules that
make up DNA) generates light
signal, which is recorded by
camera
Signal strength is proportional
to number of nucleotide
incorporated onto the DNA
strands

Eliminate amplification and wash
steps with zero wave guides
(Pacific BioSciences)
http://www.youtube.com/watch?v=v8p4ph2MAvI from 1:50 to 3:50

 Uses Zero Mode Wave Guides
 They are
◦ Very small container: zepto-liters (10-21 liters, 50
nanometers in diameter)
◦ fabricated in a 100nm metal film on a silicon dioxide
substrate
◦ enough room for 600,000 molecules of liquid water
at room temperature
◦ How much smaller can they be made?

 Uses semiconductor chips to sequence DNA by
detecting PH differences between A, G, C, and T
◦ Thus, no lasers, cameras, or amplification are used
 A micro-well containing template DNA strand is filled
with single species of deoxyribonucleotide
triphosphate (dNTP)
◦ Beneath layer of micro-wells is ion sensitive layer, below which
is ISFET ion sensor.
◦ All layers are contained in CMOS semiconductor chip
◦ If the introduced dNTP is complementary to leading template
nucleotide, it is incorporated into growing strand
◦ This causes release of a hydrogen ion that triggers ISFET ion
sensor, indicating a reaction has occurred
http://www.nature.com/news/2010/101214/full/news.2010.674.html
http://en.wikipedia.org/wiki/Ion_semiconductor_sequencing
http://www.lifescientist.com.au/article/394936/feature_sequencing_3_0/?pp=2

Done in Massively Parallel For each well
Matches cause
ion to be
released
Multiple matches
cause multiple
ions to be
released
No matches
no ions are
released

 While first sequencers used older (i.e., large feature sizes)
semiconductor technology, newer ones use smaller
feature sizes and thus are faster than older ones
 http://www.youtube.com/watch?v=JHzkYDyMzOg&feature=relmfu (2:30-
4:15)
 For example, first sequencer (314) had 1.2 million wells
while most recent one (Proton II) has 660 million wells
◦ How much smaller can these wells be made?
◦ Since 256GB memory chips (1 byte = 8 bits) exist, can ion torrent
be able to provide 256 x 8 billion wells or about 2 trillion wells in
next few years?
◦ After that improvements may slow as ion torrent's improvements
depend on further reductions in feature sizes of semiconductor
technology
http://www.nature.com/news/2010/101214/full/news.2010.674.html
http://en.wikipedia.org/wiki/Ion_semiconductor_sequencing
http://www.lifescientist.com.au/article/394936/feature_sequencing_3_0/?pp=2

Squeeze DNA through a nanoscopic pore (about 1.4 nm)
in a semiconductor and read the distinctive change each
letter in the sequence makes in the amount of current
flowing through the pore
NanoPores

 DNA moves through a nano-pore at remarkably
high velocities and thus only a small number of
ions (as few as ~100) are available in the nano-
pore to correctly identify nucleotides
◦ so the small changes in the ionic current due to the
presence of different nucleotides are overwhelmed by
thermodynamic fluctuations
 Challenge is to reduce the translocation velocity
so that the ions can be correctly identified
http://www.youtube.com/watch?v=wvclP3GySUY

http://www.nature.com/nnano/journal/v6/n10/fig_tab/nnano.2011.129_
F1.html
nt=nucleotides
Reductions in Translocation Velocity over Time

 Great for work in field, for example studying EBOLA
virus in Africa
 https://www.youtube.com/watch?v=CE4dW64x3Ts:
1:00 to 2:00
https://www.nanoporetech.com/community/minion-flow-cell-pricing
Package
Price
Number of
flow cells
Price per
flow cell
$900 1 $900.00
$9,480 12 $790.00
$16,200 24 $675.00
$24,000 48 $500.00

Personal Sequencing, Garage Biology
Sequencing can be done in your
home, office, garage, or in field
Sequence your own DNA
multiple times in your life
Sequence the DNA from a bucket
of ocean water, sewage, or
handful of dirt
Find proteins to manufacture
other things
Combined with 3D printers, PCs,
and the Internet, there is no
limit to what we can do as
individuals

 Why do Costs Fall and Other Improvements
Occur?
 New Methods Continue to Emerge
◦ Pyrosequencing (454 Life Sciences/Roche and
Illumina)
◦ Single-molecule real-time sequencing (Pacific Bio)
◦ Semiconductor arrays (Ion Torrent)
◦ Nanopores (Oxford Nanopore Technologies)
◦ Data analysis, compression, and cloud computing
 Synthesizing DNA
 Who Cares? What are the Implications?
 Conclusions

 Many believe this
will be the
bottleneck in
genome sequencing
 Partial solution:
because there are
redundancies in the
data, better
algorithms can
speed up the
sequencing
Source: Nature 498 pp. 255-260, 13 June 2013

a) File sizes of the uncompressed, compressed with links and edits, and unique sequence data
sets with default parameters. (b) Run times of BLAST, compressive BLAST and the coarse search
step of compressive BLAST on the unique data ('coarse only'). Error bars, s.d. of five runs.
Reported runtimes were on a set of 10,000 simulated queries. For queries that generate very few
hits, the coarse search time provides a lower bound on search time. (c) Run times of BLAT,
compressive BLAT and the coarse search step on the unique data ('coarse only') for 10,000

 For storage and processing
 How to encourage sharing of data?
 How to protect privacy?
 Who will be the leading providers and
users of these services?
 How will this impact on the overall
industry of health care?
◦ Might this globalize health care?

 We can synthesize new forms of DNA
 Make new drugs, crops, or materials
 Test them
 Then synthesize/design newer forms of DNA
 Keep iterating and making better drugs,
crops, and materials

The cost of synthesizing DNA is also drop
http://singularityhub.com/2012/09/17/new-software-makes-synthesizing-dna-as-easy-as-

http://www.synthesis.cc/cgi-bin/mt/mt-search.cgi?blog_id=1&tag=Carlson%20Curves&limit=20

About 5 years behind sequencing

 Most drugs are naturally occurring substances
 But improvements in our knowledge of humans
and other organisms and reductions in cost of
sequencing and synthesizing DNA increase
possibility of synthesizing drugs
◦ Begins with DNA "target”: naturally existing cellular or
molecular structure involved in pathology of interest
◦ many targets are proteins whose function has become
clear from basic scientific research
◦ Sequence protein’s DNA and then synthesize drug that
acts on this protein
 This has created a field, called Bio-informatics;
many startups are pursuing this field:
https://angel.co/bioinformatics
Gary Pisano, Science Business: The promise, the reality, and the future of biotech, Chapters 2 and 3

 If we can reduce the cost of drug development,
we can target smaller groups of people with
drugs
 How about synthesizing drugs for individuals?
 How about understanding which diseases a
human might be susceptible by sequencing their
DNA?
 Even if we cannot synthesize drugs for
individuals, can we better assign drugs to
individuals by better understanding which
humans are susceptible to known side effects
 23andMe is targeting this market
Gary Pisano, Science Business: The promise, the reality,
and the future of biotech, Chapters 2 and 3

 Scientists have recognized about 6,000 diseases
that can be traced to one or more genes
 Gleevec treats myeloid leukemia
◦ Blocks activity of protein BCR-ABL; it comes from abnormal
gene created by a merge of chromosomes 9 and 22
 Crizitnonib treats lung cancer
◦ mutated version of gene called ALK, encodes protein that
instructs lung cells to divide uncontrollably
 Vemurafenib treats melanoma
◦ Attacks protein that is generated by mutated version of a
gene called BRAF
 Problems
◦ Many cancers driven by more than one mutation and genes
involved in repair are often involved with mutations,
$100,000 for 4 doses of one drug
Source: Getting Close and Personal, Economist, January 4, 2014. The age of the red pen, Economist, August 22, 2015
http://www.economist.com/news/briefing/21661799-it-now-easy-edit-genomes-plants-animals-and-humans-age-red-pen

 Variants that
 1. codes for extra-strong bones (LRP5 G171V/+).
 2. codes for lean muscles (MSTN).
 3. makes people less sensitive to pain — something that could be
dangerous, as pain can be a useful warning signal, but may be helpful
in some contexts (SCN9A).
 4. associated with low odor production(ABCC11).
 5. makes people more resistant to viruses (CCR5, FUT2).
 6. connected to a low risk of coronary disease(PCSK9).
 7. associated with a low risk of Alzheimer’s disease (APP A63T/+).
 8. associated with a low cancer risk (GHR, GH).
 9. associated with a low risk of type 2 diabetes(SLC30A8).
 10. associated with a low risk of type 1 diabetes(IFIH1 E627X/+).
 Or is this playing God?
http://www.businessinsider.sg/gene-edits-to-make-you-stronger-and-healthier-2015-4/#ixzz3jko9ehuQ

 Better sensors (cameras,
infrared, fluorescence, lasers)
and mechanical controls
enable complete control and
measurement over crop
growth
 DNA sequencing and DNA
synthesizing enable
characterization and
replication of high
performing crops (sometimes
called GMO)
 Other biological materials
http://www.aber.ac.uk/en/media/departmental/ibers/facilities/phenomicscentre/BBC-FOCUS-NPPC-Feature.pdf
 Cellulosic ethanol
 Algae: http://www.slideshare.net/Funk98/presentations

Source: https://www.soils.org/publications/cs/articles/46/2/528
Improvements in U.S. Corn Yields through New Seeds

Improvements in Yield for other Crops
U.S. Department of Agriculture and Michael Bomford, Crop Yield Projections

 U.S. Food and Drug
Administration approved
genetically engineered
salmon for consumption
 Developed by AquaBounty
Technologies, that first
approached FDA in 1990s
 Genetic modifications enable
it to grow to market size
faster, in as little as half the
time
 Will be in stores by 2017
http://www.nytimes.com/2015/11/20/business/geneticall
y-engineered-salmon-approved-for-consumption.html

 Food can be expensive (usually smaller fish),
particularly if fish are to have the right oils
 Geneticists have added pertinent genes to
oil-rich plants to make better food
 Successful tests in green house as did
outdoor tests, led to oil-rich plants
 Added benefits of these plants
◦ Less build up of mercury in fish
◦ Big problem with fish raised on smaller fish
(common method of raising fish)
◦ Consumers concerned with mercury in fish also
often dislike genetically modified food
Something Fishy, Economist, July 11, 2015, p. 69

Bio-Fuels: Scale-up of cellulosic ethanol
production might enable economic feasibility,
But new plant sources for bio-fuels also needed

 It’s not just about
◦ making bio-fuels from the non-food part of the
plant or
◦ scaling up the production in order to reduce cost
 It’s also about Developing Better Organisms
◦ Better cellulose that produces more ethanol per
weight, while still enabling the plant to produce lots
of food
◦ Better algae that consumes more carbon dioxide
and generates more energy per weight or area
http://www.theguardian.com/science/2012/jan/14/synthetic-biology-spider-goat-genetics

 For example, spider silk is very strong
 But difficult to harvest spider silk, partly because
it is hard to raise spiders (they eat each other)
 Scientists introduced the gene for spider silk into
goats so spider silk would be produced in their
milk
 Now spider silk is produced in the goat’s milk
and scientists are trying to improve the results
 It is expected that many other natural substances
can be manufactured in this way
http://www.theguardian.com/science/2012/jan/14/synthetic-biology-spider-goat-genetics

 Enzymes, plastics, textiles, dyes
 Many of these are now made from fossil
fuels but were once made form natural
substances
 Can we return to biological feedstocks?
◦ Modify yeast so that sugar can be turned into
useful compounds such as malaria drugs and
biofuels
◦ Bring a switch from fossil fuels to biological
feedstocks such as sugar, starch, and cellulose

 Registry of Standard Biological Parts
◦ More than 10,000 parts
 Can build complex systems from these
parts
 Genetically Engineered Machine
Competition
◦ Students compete to build complex systems
◦ One group built a biological light detector with a
resolution of 100 million pixels per square inch
 Will biological systems ever compete with
electronic systems?

 Build complex systems from simple parts in
small decentralized labs
 Use simplified version of DNA sequencers,
called PCR (polymerase chain reaction), to
identify a specific segment of DNA
◦ Costs have fallen to $500
 Other technologies support use of PCR
◦ Autodesk develops design tools for DNA
◦ Fluid handling robots from Opus
◦ 3D printer for living things
Bio-hackers of the world, unite; Economist September 6, 2014

 DNA synthesizing equipment can be used to
make (and replicate) DNA
 One challenge is how to insert DNA into a cell,
so that the cell can then replicate itself
◦ Each cell contains DNA needed for a specific organism
◦ Each cell may even contain the DNA for features that no longer
exist and the features can be turned back on
 First done by Craig Venter’s team in May 2010
◦ His team synthesized an entire bacterial genome and
“took over” a cell by inserting the DNA into the cell
 Can this be done for more complex life forms?
Source: Michio Kaku, Physics of the Future: How Science Will Shape
Human Destiny and Our Daily Lives by the Year 2100 (2011)

More Complex Organisms Require More Base Pairs and
thus more years for their Synthesizing

 The cost of sequencing and
synthesizing DNA continues to fall
 A major reason for the cost reductions
is the benefits from reductions in scale
◦ Similar to those in ICs, bio-electronic ICs,
and MEMS
◦ A powerful way to reduce costs
 Further reductions in scale and thus
further cost reductions appear possible

 If costs continue to fall, low cost and small
DNA sequencers and synthesizers will change
drug discovery, health care, and science
◦ How will we do drug discovery in the future?
 What kind of analyses can help us understand
how these trends will change drug discovery
and health care?
 What kinds of opportunities will emerge for
firms as vast amounts of data become
available for analysis?

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