Sequencing and analysis of gene expression
Jiří Fajkus
CG080 – Methods of Genomics and Proteomics

220px-RNA-codons 220px-RNA_chemical_structure 500px-Aminoacids_table



•
•A = adenine
•C = cytosine
•G = guanine
•T = thymine
•R = G A (purine)
•Y = T C (pyrimidine)
•K = G T (keto)
•M = A C (amino)
•S = G C (strong bonds)
•W = A T (weak bonds)
•B = G T C (all but A)
•D = G A T (all but C)
•H = A C T (all but G)
•V = G C A (all but T)
•N = A G C T (any)
•
•http://www.wiley.com/college/pratt/0471393878/student/animations/dna_sequencing/index.html

220px-Frederick_Sanger2
•Nobel Prize in Chemistry (1958) – amino acid sequence of insulin
Nobel Prize in Chemistry (1980) – dideoxy method of DNA sequencing
•Frederick Sanger (13. 8. 1918 – 19. 11. 2013)
•Sanger F, Nicklen S, Coulson AR (December 1977). "DNA sequencing with chain-terminating
inhibitors". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5463–7.

•Historical methods:
•1975 - Sanger and Coulson – plus-minus sequencing (4 pairs of plus (1 dNTP) and minus (3 other
dNTPs) reactions, restriction enzyme fragments as primers for elongation by DNA polymerase I.
• – used for sequencing of 5,386 nucleotides of the single-stranded bacteriophage φX174
•Chemical sequencing / Maxam-Gilbert Sequencing (Nobel Prize 1980)
•1976–1977- Allan Maxam and Walter Gilbert  (Maxam AM, Gilbert W (Feb 1977). "A new method for
sequencing DNA". Proc. Natl. Acad. Sci. U.S.A. 74 (2): 560–4).
•
•1) radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma-32P
ATP) and purification of the DNA fragment to be sequenced.
•2) Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide
bases in each of four reactions (G, A+G, C, C+T).
•purines (A+G) are depurinated using formic acid,
•guanines (G) (and to some extent the adenines) are methylated by dimethyl sulfate
•pyrimidines (C+T) – bases are cleaved by hydrazine.
•The addition of salt (NaCl) to the hydrazine reaction inhibits the methylation of thymine for the
C-only reaction.
•3) The modified DNAs are then cleaved by hot piperidine at the position of the modified base. Thus
a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in
each molecule.
•4) The fragments in the four reactions are electrophoresed side by side in denaturing PAGE for
size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography,
yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the
sequence may be inferred.




Maxam
•
•
•
ChemSeq 1-s2 2_1_5c




•Chain-termination sequencing (Sanger sequencing, dideoxy-sequencing)
- the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.
- Requirements: ss (heat-denatured) DNA template, a DNA primer, a DNA polymerase, normal
deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs, ddNTPs) that terminate
DNA strand elongation.
-The DNA sample is divided into 4 separate sequencing reactions, containing all four of the
standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is
added only one of the four ddNTPs (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating
nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two
nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying
length.
- The newly synthesized and labelled DNA fragments are heat denatured, and separated by size (with
a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel
with each of the four reactions run in one of four individual lanes (lanes A, T, G, C);
- the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be
directly read off the X-ray film or gel image.

Sequencing
-In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA
fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result
of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP).
The relative positions of the different bands among the four lanes are then used to read (from
bottom to top) the DNA sequence.
dnaseq1
•One of the four sequencing reactions

File:DNA Sequencin 3 labeling methods.jpg File:CE Basic.jpg



File:Radioactive Fluorescent Seq.jpg



File:Sanger sequencing read display.gif



File:DNA Sequencing gDNA libraries.jpg
•General sequencing strategy


•Next generation sequencing
star-trek-tng1


•Pyrosequencing (Ronaghi and Nyrén, 1996)
•A parallelized version of pyrosequencing was developed by 454 Life Sciences, (now part of Roche
Diagnostics) – 454 Pyrosequencing.
•Amplification of DNA inside water droplets in an oil solution (emulsion PCR), with each droplet
containing a single DNA template attached to a single primer-coated bead that then forms a clonal
colony. The sequencing machine contains many picolitre-volume wells each containing a single bead
and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the
individual nucleotides added to the nascent DNA, and the combined data are used to generate
sequence read-outs. This technology provides intermediate read length and price per base compared
to Sanger sequencing on one end and Solexa and SOLiD on the other.
Sequencing1_02
•see ANIMATION:
http://www.roche-applied-science.com/publications/multimedia/genome_sequencer/presentation/wbt.htm

Fragments_05
•The Roche 454/GS FLX Sequencing Technology
•
•The GS FLX sequencer supports sequencing of various different nucleic acid starting materials such
as genomic DNA, PCR products, BACs and cDNA. Samples consisting of longer sequences are
first sheared into a random library of 300-800 base-pair long fragments.
•Adaptors essential for purification, amplification and sequencing are added to both ends of the
fragments. If the sample is double stranded one strand is removed and the remaining single strandes
are used in the following steps.
SS_05 Bead_02
•Aided by the adaptors individual fragments are captured on their own unique beads. A bead and the
bound fragment together with a water-in-oil emulsion form a microreactor so that each fragment can
be amplified without contamination via the so called emulsion PCR (emPCR). The entire fragment
collection is amplified in parallel.

BeadPolySeq_02
•The emPCR amplifies each fragment several million times. After amplification the emulsion shell is
broken and the clonally amplified beads are ready for loading onto the fibre-optic PicoTiterDevice
for sequencing.
Wells_02
•The PicoTiterPlate is loaded with one fragment carrying bead per well and smaller beads with the
enzymes necessary for sequencing.
•Sequencing is accomplished by synthesizing the complementary strands of the bead attached
templates. In a number of cycles the four bases (ATGC) are sequentially washed over the
PicoTiterPlate. The incorporation of a new base is associated with the release of inorganic
pyrophosphate starting a chemical cascade. This results in the generation of a light signal which
is captured by a CCD camera.
Sequencing1_02

Pyrosequencing technology step3.gif
•ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP
drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light
in amounts that are proportional to the amount of ATP. The light produced in the
luciferase-catalyzed reaction is detected by a charge coupled device (CCD) chip and seen as a peak
in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the
number of nucleotides incorporated.
•Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alfa-thio
triphosphate (dATP·S) is used as a substitute for the natural dATP since it is efficiently used by
the DNA polymerase, but not recognized by the luciferase. As the process continues, the
complementary DNA strand is built up and the nucleotide sequence is determined from the signal
peaks in the Pyrogram trace.
Pyrosequencing technology step5.gif




•llumina (Solexa) sequencing
•
•Solexa, now part of Illumina, developed a sequencing technology based on reversible
dye-terminators.
•DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies
are formed (isothermal bridge amplification). Four types of reversible terminator bases (RT-bases)
are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can
only be extended one nucleotide at a time. A camera takes images of the fluorescently labelled
nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA,
allowing the next cycle.
•SEE ANIMATION:
http://www.illumina.com/media/flash_player.ilmn?dirname=systems&swfname=GA_workflow_vid&width=780&h
eight=485&iframe

•llumina



•llumina



•llumina



•llumina



•SOLiD sequencing
•
•Applied Biosystems' SOLiD technology employs sequencing by ligation. Here, a pool of all possible
oligonucleotides of a fixed length are labeled according to the sequenced position.
Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching
sequences results in a signal informative of the nucleotide at that position. Before sequencing,
the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same
DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths
comparable to Illumina sequencing.
•SEE ANIMATION: http://www.youtube.com/watch?v=nlvyF8bFDwM

abi-fig5
•SOLiD sequencing
AT

http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-techn
ology/solid-next-generation-sequencing/data-images.Par.33471.Image.-1.0.1.gif
Library Preparation
Prepare one of the two types of libraries for SOLiD™ System sequencing-fragment or mate-paired.
Your choice of library depends on the application you're performing and the information you desire
from your experiments.
•SOLiD sequencing

http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-techn
ology/solid-next-generation-sequencing/data-images.Par.62718.Image.-1.0.1.gif
Emulsion PCR/Bead Enrichment
Prepare clonal bead populations in microreactors containing template, PCR reaction components,
beads, and primers.
After PCR, denature the templates and perform bead enrichment to separate beads with extended
templates from undesired beads. The template on the selected beads undergoes a 3’ modification to
allow covalent attachment to the slide.
•SOLiD sequencing

http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-techn
ology/solid-next-generation-sequencing/data-images.Par.67475.Image.-1.0.1.gif
Bead Deposition
Deposit 3’ modified beads onto a glass slide. During bead loading, deposition chambers enable you
to segment a slide into one, four, or eight sections. A key advantage of the system is the ability
to accommodate increasing densities of beads per slide, resulting in a higher level of throughput
from the same system.
•SOLiD sequencing

data-images
Sequencing by Ligation
1. Primers hybridize to the P1 adapter sequence on the templated beads.
2. A set of four fluorescently labeled di-base probes compete for ligation to the sequencing
primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in
each ligation reaction.
3. Multiple cycles of ligation, detection and cleavage are performed with the number of cycles
determining the eventual read length.
4. Following a series of ligation cycles, the extension product is removed and the template is
reset with a primer complementary to the n-1 position for a second round of ligation cycles.

http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-techn
ology/solid-next-generation-sequencing/data-images.Par.57418.Image.-1.0.1.gif
Primer Reset
Five rounds of primer reset are completed for each sequence tag. Through the primer reset process,
virtually every base is interrogated in two independent ligation reactions by two different
primers.
For example, the base at read position 5 is assayed by primer number 2 in ligation cycle 2 and by
primer number 3 in ligation cycle 1. This dual interrogation is fundamental to the unmatched
accuracy characterized by the SOLiD™ System.
•SOLiD sequencing

a | A four-colour sequencing by ligation method using Life/APG's support oligonucleotide ligation
detection (SOLiD) platform is shown. Upon the annealing of a universal primer, a library of
1,2-probes is added. Unlike polymerization, the ligation of a probe to the primer can be performed
bi-directionally from either its 5′-PO4 or 3′-OH end. Appropriate conditions enable the selective
hybridization and ligation of probes to complementary positions. Following four-colour imaging, the
ligated 1,2-probes are chemically cleaved with silver ions to generate a 5′-PO4 group. The SOLiD
cycle is repeated nine more times. The extended primer is then stripped and four more ligation
rounds are performed, each with ten ligation cycles. The 1,2-probes are designed to interrogate the
first (x) and second (y) positions adjacent to the hybridized primer, such that the 16
dinucleotides are encoded by four dyes (coloured stars). The probes also contain inosine bases (z)
to reduce the complexity of the 1,2-probe library and a phosphorothiolate linkage between the fifth
and six nucleotides of the probe sequence, which is cleaved with silver ions106. Other cleavable
probe designs include RNA nucleotides107, 108 and internucleosidic phosphoramidates107, which are
cleaved by ribonucleases and acid, respectively. b | A two-base encoding scheme in which four
dinucleotide sequences are associated with one colour (for example, AA, CC, GG and TT are coded
with a blue dye). Each template base is interrogated twice and compiled into a string of
colour-space data bits. The colour-space reads are aligned to a colour-space reference sequence to
decode the DNA sequence. c | Pyrosequencing using Roche/454's Titanium platform. Following loading
of the DNA-amplified beads into individual PicoTiterPlate (PTP) wells, additional beads, coupled
with sulphurylase and luciferase, are added. In this example, a single type of
2′-deoxyribonucleoside triphosphate (dNTP) — cytosine — is shown flowing across the PTP wells. The
fibre-optic slide is mounted in a flow chamber, enabling the delivery of sequencing reagents to the
bead-packed wells. The underneath of the fibre-optic slide is directly attached to a
high-resolution charge-coupled device (CCD) camera, which allows detection of the light generated
from each PTP well undergoing the pyrosequencing reaction. d | The light generated by the enzymatic
cascade is recorded as a series of peaks called a flowgram. PPi, inorganic pyrophosphate.

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•Applications
•De novo sequencing: 454 (longer reads and high coverage - preferred for genome assembly)
•Resequencing: Solexa, SOLiD (shorter, but reliable reads) – identification of differences compared
to reference sequence – e.g. molecular diagnostics,...
•
•Transcriptome sequencing – substitutes for cDNA/RNA microarrays,
•- representation of reads transcripts (normalized to reference transcript) reflects abundance of
the transcript
•- more precise than microarrays
-possibility of detection of unknown transcripts
•Whole genome sequencing, 1000 genome project (human genome variation)
•Metagenomics – analysis of complete genetic material from environmental samples without
cultivation – real diversity (e.g. microbial composition of certain habitats, human organs - only
about 1 out of 10 cells in the human body is actually a human cell: most of the cells that make up
our bodies are microbes!)
•Epigenetics / epigenomics - DNA methylation, ChIP-Seq

•Ion semiconductor sequencing
•
•Ion Torrent Systems Inc. (now owned by Life Technologies) developed a system based on using
standard sequencing chemistry, but with a novel, semiconductor based detection system. This method
of sequencing is based on the detection of hydrogen ions that are released during the
polymerisation of DNA, as opposed to the optical methods used in other sequencing systems. A
microwell containing a template DNA strand to be sequenced is flooded with a single type of
nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is
incorporated into the growing complementary strand. This causes the release of a hydrogen ion that
triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer
repeats are present in the template sequence multiple nucleotides will be incorporated in a single
cycle. This leads to a corresponding number of released hydrogens and a proportionally higher
electronic signal.
•http://www.iontorrent.com/

Ion-Torrent
•In nature, when a nucleotide is incorporated into a strand of DNA by a polymerase, a hydrogen ion
is released as a byproduct.
•Step One
•Ion Torrent
cm400726f1_online

Ion-Torrent
•Step Two
•
•Ion Torrent™ uses a high-density array of micro-machined wells to perform this biochemical process
in a massively parallel way. Each well holds a different DNA template. Beneath the wells is an
ion-sensitive layer and beneath that a proprietary Ion sensor.
•Ion Torrent

Ion-Torrent
•Step Three
•If a nucleotide, for example a C, is added to a DNA template and is then incorporated into a
strand of DNA, a hydrogen ion will be released. The charge from that ion will change the pH of the
solution, which can be detected by our proprietary ion sensor. The sequencer - essentially the
world's smallest solid-state pH meter - will call the base, going directly from chemical
information to digital information.
•Ion Torrent

Ion-Torrent
•The Ion Personal Genome Machine™ (PGM™) sequencer then sequentially floods the chip with one
nucleotide after another. If the next nucleotide that floods the chip is not a match, no voltage
change will be recorded and no base will be called.
•Step Four
•Ion Torrent

Ion-Torrent
•Step Five
•If there are two identical bases on the DNA strand, the voltage will be double, and the chip will
record two identical bases called. Because this is direct detection - no scanning, no cameras, no
light - each nucleotide incorporation is recorded in seconds.
•The semiconductor will inevitably transform the life sciences, just as it has transformed every
other industry it has touched. By creating a direct connection between chemical and digital
information, Ion Torrent™ will democratize research, providing a fast, simple, scalable sequencing
solution that every lab can afford. Eventually, Ion Torrent Technology™ will also be able to
provide diagnostics that are less expensive and more reliable, improving human health around the
world.
•Ion Torrent

•HelioscopeTM single molecule sequencing
•
•Based on "true single molecule sequencing" technology, Helioscope sequencing uses DNA fragments
with added polyA tail adapters, which are attached to the flow cell surface. The next steps involve
extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled
nucleotides (one nucleotide type at a time, as with the Sanger method). The reads are performed by
the Helioscope sequencer. The reads are short, up to 55 bases per run, but recent improvement of
the methodology allows more accurate reads of homopolymers (stretches of one type of nucleotides)
and RNA sequencing.
•
•Advantages: High number of reads, no sequencing bias (no amplification step)

a | The four-colour cyclic reversible termination (CRT) method uses Illumina/Solexa's
3′-O-azidomethyl reversible terminator chemistry23, 101 (Box 1) using solid-phase-amplified
template clusters (FIG. 1b, shown as single templates for illustrative purposes). Following
imaging, a cleavage step removes the fluorescent dyes and regenerates the 3′-OH group using the
reducing agent tris(2-carboxyethyl)phosphine (TCEP)23. b | The four-colour images highlight the
sequencing data from two clonally amplified templates. c | Unlike Illumina/Solexa's terminators,
the Helicos Virtual Terminators33 are labelled with the same dye and dispensed individually in a
predetermined order, analogous to a single-nucleotide addition method. Following total internal
reflection fluorescence imaging, a cleavage step removes the fluorescent dye and inhibitory groups
using TCEP to permit the addition of the next Cy5-2′-deoxyribonucleoside triphosphate (dNTP)
analogue. The free sulphhydryl groups are then capped with iodoacetamide before the next nucleotide
addition33 (step not shown). d | The one-colour images highlight the sequencing data from two
single-molecule templates.

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•How tSMS Works
•Within two flow cells, billions of single molecules of sample DNA are captured on an
application-specific proprietary surface. These captured strands serve as templates for the
sequencing-by-synthesis process:
•1. Polymerase and one fluorescently labeled nucleotide (C, G, A or T) are added.
•2. The polymerase catalyzes the sequence-specific incorporation of fluorescent nucleotides into
nascent complementary strands on all the templates.
•3. After a wash step, which removes all free nucleotides, the incorporated nucleotides are imaged
and their positions recorded.
•4. The fluorescent group is removed in a highly efficient cleavage process, leaving behind the
incorporated nucleotide.
•5. The process continues through each of the other three bases.
•Multiple four-base cycles result in complementary strands greater than 25 bases in length
synthesized on billions of templates—providing a greater than 25-base read from each of those
individual templates.
•http://www.helicosbio.com/Technology/TrueSingleMoleculeSequencing/tabid/64/Default.aspx




•Single molecule SMRTTM sequencing
•
•SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in
zero-mode wave-guides (ZMWs) - small well-like containers with the capturing tools located at the
bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the
ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The wells are
constructed in a way that only the fluorescence occurring by the bottom of the well is detected.
The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand,
leaving an unmodified DNA strand. According to Pacific Biosciences, the SMRT technology developer,
this methodology allows detection of nucleotide modifications (such as cytosine methylation). This
happens through the observation of polymerase kinetics. This approach allows reads of up to 15,000
nucleotides, with mean read lengths of 2.5 to 2.9 kilobases

smrt_Technology
•The SMRT Cell
One of the fundamental challenges with observing a DNA polymerase working in real time is the
ability to detect the incorporation of a single nucleotide, taken from a large pool of potential
nucleotides, during DNA synthesis. We applied the same principle that operates in the metallic
screen of a microwave oven door. In a microwave oven, the screen is perforated with holes that are
much smaller than the wavelength of the microwaves. Because of their relative size, the holes
prevent the much longer microwaves from passing through and penetrating the glass. However, the
much smaller wavelength visible light is able to pass through the holes in the screen, allowing
food to be visible. We have reduced this same principle to the nanoscale and we call our innovation
a zero-mode waveguide, or ZMW.
•With an active polymerase immobilized at the bottom of each ZMW, nucleotides diffuse into the ZMW
chamber. In order to detect incorporation events and identify the base, each of the four
nucleotides A, C, G and T are labeled with a different fluorescent dye having a distinct emission
spectrum. Since the excitation illumination is directed to the bottom of the ZMW, nucleotides held
by the polymerase prior to incorporation emit an extended signal that identifies the base being
incorporated.

•A ZMW is a hole, tens of nanometers in diameter, fabricated in a 100nm metal film deposited on a
glass substrate. The small size of the ZMW prevents visible laser light, which has a wavelength of
approximately 600nm, from passing entirely through the ZMW. Rather than passing through, the light
exponentially decays as it enters the ZMW. Therefore, by shining a laser through the glass into the
ZMW, only the bottom 30nm of the ZMW becomes illuminated. Within each ZMW, a single DNA polymerase
molecule is anchored to the bottom glass surface using a proprietary technique. Nucleotides, each
type labeled with a different colored fluorophore, are then flooded above an array of ZMWs at the
required concentration. Diffusion at the nanoscale is incredibly fast. Within microseconds, labeled
nucleotides travel down into the ZMW, surround the DNA polymerase, then diffuse back up and exit
the hole. As no laser light penetrates up through the holes to excite the fluorescent labels, the
labeled nucleotides above the ZMWs are dark. Only when they diffuse through the bottom 30nm of the
ZMW do they fluoresce. When the correct nucleotide is detected by the polymerase, it is
incorporated into the growing DNA strand in a process that takes milliseconds in contrast to simple
diffusion which takes microseconds. This difference in time results in higher signal intensity for
incorporated versus unincorporated nucleotides, which creates a high signal-to-noise ratio. Thus,
the ZMW has the ability to detect a single incorporation event against the background of
fluorescently labeled nucleotides at biologically relevant concentrations.
smrt_Technology
•DNA sequencing is performed on proprietary SMRT Cells, each having an array of approximately
75,000 ZMWs. Each ZMW is capable of containing a DNA polymerase loaded with a different strand of
DNA sample. As a result, the SMRT Cell enables the potential detection of approximately 75,000
single molecule sequencing reactions in parallel.

•Phospholinked Nucleotides
Previous labeling technologies attach a fluorescent label to the base of the nucleotide, which is
incorporated into the DNA strand. This is problematic for any system to observe DNA synthesis in
real time because the dye’s large size can interfere with the activity of the DNA polymerase.
Typically, a DNA polymerase can incorporate only a few base-labeled nucleotides before it halts.
Our proprietary phospholinked nucleotides have a fluorescent dye attached to the phosphate chain of
the nucleotide rather than to the base. The phosphate chain is cleaved when the nucleotide is
incorporated into the DNA. Thus, upon incorporation of a phospholinked nucleotide, the DNA
polymerase naturally frees the dye molecule from the nucleotide when it cleaves the phosphate
chain. Upon cleaving, the label quickly diffuses away, with no evidence of labeling remaining.

•21.3.2017
> >
Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of a single ZMW
nanostructure containing a single DNA polymerase (Φ29) bound to the bottom glass surface. The ZMW
and the confocal imaging system allow fluorescence detection only at the bottom surface of each
ZMW. (B) Representation of fluorescently labeled nucleotide substrate incorporation on to a
sequencing template. The corresponding temporal fluorescence detection with respect to each of the
five incorporation steps is shown below. Reprinted with permission from ref 39. Copyright 2009
American Association for the Advancement of Science.
Published in: Thomas P. Niedringhaus; Denitsa Milanova; Matthew B. Kerby; Michael P. Snyder;
Annelise E. Barron; Anal. Chem.  2011, 83, 4327-4341.
DOI: 10.1021/ac2010857
Copyright © 2011 American Chemical Society

•
•Michal  Levy-Sakin , Yuval  Ebenstein
• Beyond sequencing: optical mapping of DNA in the age of nanotechnology and nanoscopy
•Current Opinion in Biotechnology, Volume 24, Issue 4, 2013, 690 - 698
•Individual DNA molecules hundreds of kbp long may be stretched and visualized by optical
microscopy.
•► An optical barcode is generated by fluorescent labeling of short sequence motifs along the
stretched DNA.
•► Optical maps complement DNA sequencing for gap closing, finishing, validation and de novo
assembly of genomes.
•► Genome structural variations not accessible to sequencing or DNA arrays may be directly
visualized.
•► Epigenetic marks such as DNA methylation and DNA binding proteins may also be mapped on single
genomic fragments.

•Genomic data domains — from whole chormosome to single nucleotide. (a) Spectral human karyotype
illustration (taken by Jane Ades, NHGRI, adapted from www.genome.gov). Spectral Karyotyping (SKY)
and FISH analysis allow the detection of whole or parts of chromosomes in resolution ranging
between a few Mbp to 1 Gbp. (b) Optical mapping allows detection of DNA with resolution of 1 kbp to
several Mbp, here as an example for optical mapping, genomic morse code of Herpes simplex virus-1
(HSV-1) isomers (modified from [ 44], with permission, American Society for Microbiology).
Fluorescent hybridization arrays illustrating the HSV isomers P and IL are shown below the scheme.
The sizes of the fragments shown are given in kbp according to the reference HSV-1 genome sequence
(Genbank accession number: NC_001806). (c) The highest resolution of DNA mapping is achieved by DNA
sequencing which allows single nucleotide analysis.
•Michal  Levy-Sakin , Yuval  Ebenstein
• Beyond sequencing: optical mapping of DNA in the age of nanotechnology and nanoscopy
•Current Opinion in Biotechnology, Volume 24, Issue 4, 2013, 690 - 698

•Optical mapping schemes. (a) Scanning electron microscope (SEM) image of a highly parallel
nanochannel-array chip (source: BioNano Genomics, with permission). (b) BAC DNA molecules (green)
are nicked by an enzyme at specific sequence motifs and repaired by a polymerase that incorporates
fluorescently labeled nucleotides (orange dots) and imaged in nanochannel-array. (c) DNA molecular
combing. Large rearrangements in BRCA1 are detected in breast cancer patients. Del ex 8–13 is
visible as a deletion of the blue signal S7B1. (d) Restriction-optical mapping. A 90 kbp insertion
is detected (red arrows). Included below the map is a montage of several of the single-molecule
images that give evidence to support this insertion. (e) Future perspective for optical mapping in
nanochannels: DNA molecules (green) are specifically labeled with different colored probes
revealing different genomic/epigenomic information such as sequence motifs and methylation sites
(colored dots). An applied electric field drives the molecules through a series of progressively
smaller nanoscale obstacles (gray circles) that funnel the molecules into nanochannels. Once DNA is
stretched and confined within the channels, the distances between labels can be accurately measured
using a fluorescence microscope. Software is used to generate a consensus map of the sequence
motifs overlaid with the epigenetic patterns of individual molecules. The maps facilitate the
analysis of structural and epigenetic variation, such as sequence duplications and methylation
patterns.
•Michal  Levy-Sakin , Yuval  Ebenstein
• Beyond sequencing: optical mapping of DNA in the age of nanotechnology and nanoscopy
•Current Opinion in Biotechnology, Volume 24, Issue 4, 2013, 690 - 698

•Beyond the sequence — optical mapping of chromatin. (a) Schematic illustration of DNA labeling.
DNA sequences, chromatin modifications and epigenetic data such as methylation can be detected by
optical mapping. (b) Cropped color overlay images of T7 genomes (white) carrying sequence specific
tags (red) and fluorescently labeled RNAP (green). Scale bar 3 μm. (c) Stretching of single
chromatin fragments characterized by atomic force microscopy (AFM) (first panel) and fluorescence
microscopy (second and third panels). Three-dimensional representation of the chromatin molecules
reveals nucleosome distribution and positioning along the stretched chromatin molecule. Chromatin
fragments were stained with YOYO-1 (green) and labeled with Alexa647-histone H3 probes (red).
•Michal  Levy-Sakin , Yuval  Ebenstein
• Beyond sequencing: optical mapping of DNA in the age of nanotechnology and nanoscopy
•Current Opinion in Biotechnology, Volume 24, Issue 4, 2013, 690 - 698

•Nanopore DNA sequencing
•
•This method is based on the readout of electrical signal occurring at nucleotides passing by
alpha-hemolysin pores covalently bound with cyclodextrin. The DNA passing through the nanopore
changes its ion current. This change is dependent on the shape, size and length of the DNA
sequence. Each type of the nucleotide blocks the ion flow through the pore for a different period
of time.
•http://www.nanoporetech.com/technology/analytes-and-applications-dna-rna-proteins/dna-an-introduct
ion-to-nanopore-sequencing
Nanopore_sensing_101_0_rs

Nanopore_sensing_101_0_rs
•This diagram shows a protein nanopore set in an electrically resistant membrane bilayer.  An ionic
current is passed through the nanopore by setting a voltage across this membrane.

If an analyte passes through the pore or near its aperture, this event creates a characteristic
disruption in current. By measuring that current it is possible to identify the molecule in
question. For example, this system can be used to distinguish the four standard DNA bases and G, A,
T and C, and also modified bases.  It can be used to identify target proteins, small molecules, or
to gain rich molecular information for example to distinguish the enantiomers of ibuprofen or
molecular binding dynamics.
•Strand sequencing
•Exonuclease sequencing

•21.3.2017
> >
Nanopore DNA sequencing using electronic measurements and optical readout as detection methods. (A)
In electronic nanopore schemes, signal is obtained through ionic current, tunneling current, and
voltage difference measurements. Each method must produce a characteristic signal to differentiate
the four DNA bases. (B) In the optical readout nanopore design, each nucleotide is converted to a
preset oligonucleotide sequence and hybridized with labeled markers that are detected during
translocation of the DNA fragment through the nanopore.
Published in: Thomas P. Niedringhaus; Denitsa Milanova; Matthew B. Kerby; Michael P. Snyder;
Annelise E. Barron; Anal. Chem.  2011, 83, 4327-4341.
DOI: 10.1021/ac2010857
Copyright © 2011 American Chemical Society

> >
Biological nanopore scheme employed by Oxford Nanopore. (A) Schematic of αHL protein nanopore
mutant depicting the positions of the cyclodextrin (at residue 135) and glutamines (at residue
139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the
arginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is
attached to the top of the nanopore to cleave single nucleotides from the target DNA strand and
pass them through the nanopore. (D) A residual current-vs-time signal trace from an αHL protein
nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E)
Strand sequencing: ssDNA is threaded through a protein nanopore and individual bases are
identified, as the strand remains intact.
Published in: Thomas P. Niedringhaus; Denitsa Milanova; Matthew B. Kerby; Michael P. Snyder;
Annelise E. Barron; Anal. Chem.  2011, 83, 4327-4341.
DOI: 10.1021/ac2010857
Copyright © 2011 American Chemical Society

•21.3.2017
> >
Several synthetic nanopore sequencing device designs. (A) The device consists of 1–5 nm thick
graphene membrane which is suspended in a Si chip coated with 5 μm SiO2 layer. It is placed in a
PDMS cell with microfluidic channels on both sides of the chip. (B) A nanopore (shown in the inset
to the figure) is drilled through a graphene membrane, which is suspended in SiNx across a Si
frame. The graphene membrane separates two ionic solutions and is in contact with Ag/AgCl
electrodes. (C) IBM DNA transistor setup. A nanometer sized pore is fabricated using an electron
beam. Electric field is created between the gated regions allowing for charge trapping. The
substrate is composed of metal and dielectric regions, labeled with ‘‘M’’ and ‘‘D’’, respectively.
(D) HANS method adopted by NABsys for electronic readout of DNA fragments through solid-state
nanopores. 6-mer oligonucleotide probes are hybridized to ssDNA fragments, and current-verses-time
trace is detected.
Published in: Thomas P. Niedringhaus; Denitsa Milanova; Matthew B. Kerby; Michael P. Snyder;
Annelise E. Barron; Anal. Chem.  2011, 83, 4327-4341.
DOI: 10.1021/ac2010857
Copyright © 2011 American Chemical Society

•VisiGen Biotechnologies approach
•
•VisiGen Biotechnologies introduced a specially engineered DNA polymerase for use in their
sequencing. This polymerase acts as a sensor - having incorporated a donor fluorescent dye by its
active centre. This donor dye acts by FRET (fluorescent resonance energy transfer), inducing
fluorescence of differently labeled nucleotides. This approach allows reads performed at the speed
at which polymerase incorporates nucleotides into the sequence (several hundred per second). The
nucleotide fluorochrome is released after the incorporation into the DNA strand. The expected read
lengths in this approach should reach 1000 nucleotides.

•Future methods
•Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A single pool of
DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array
containing known sequences. Strong hybridization signals from a given spot on the array identifies
its sequence in the DNA being sequenced. Mass spectrometry may be used to determine mass
differences between DNA fragments produced in chain-termination reactions.
•DNA sequencing methods currently under development include labeling the DNA polymerase, reading
the sequence as a DNA strand transits through nanopores,and microscopy-based techniques, such as
AFM or transmission electron microscopy that are used to identify the positions of individual
nucleotides within long DNA fragments (>5,000 bp) by nucleotide labeling with heavier elements
(e.g., halogens) for visual detection and recording. Third generation technologies aim to increase
throughput and decrease the time to result and cost by eliminating the need for excessive reagents
and harnessing the processivity of DNA polymerase.
•In microfluidic Sanger sequencing the entire thermocycling amplification of DNA fragments as well
as their separation by electrophoresis is done on a single glass wafer (approximately 10 cm in
diameter) thus reducing the reagent usage as well as cost. In some instances researchers have shown
that they can increase the throughput of conventional sequencing through the use of microchips.
Research will still need to be done in order to make this use of technology effective.
•In October 2006, the X Prize Foundation established an initiative to promote the development of
full genome sequencing technologies, called the Archon X Prize, intending to award $10 million to
"the first Team that can build a device and use it to sequence 100 human genomes within 10 days or
less, with an accuracy of no more than one error in every 100,000 bases sequenced, with sequences
accurately covering at least 98% of the genome, and at a recurring cost of no more than $10,000
(US) per genome."
•Each year NHGRI promotes grants for new research and developments in genomics. 2010 grants and
2011 candidates include continuing work in microfluidic, polony and base-heavy sequencing
methodologies.

•Major landmarks in DNA sequencing
•
•1953 Discovery of the structure of the DNA double helix.
•1972 Development of recombinant DNA technology, which permits isolation of defined fragments of
DNA; prior to this, the only accessible samples for sequencing were from bacteriophage or virus
DNA.
•1977 The first complete DNA genome to be sequenced is that of bacteriophage φX174.
•1977 Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation".
• Frederick Sanger, independently, publishes "DNA sequencing with chain-terminating inhibitors".
•1984 Medical Research Council scientists decipher the complete DNA sequence of the Epstein-Barr
virus, 170 kb.
•1986 Leroy E. Hood's laboratory at the California Institute of Technology and Smith announce the
first semi- automated DNA sequencing machine.
•1987 Applied Biosystems markets first automated sequencing machine, the model ABI 370.
•1990 The U.S. National Institutes of Health (NIH) begins large-scale sequencing trials on
Mycoplasma capricolum, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae (at
US$0.75/base).
•1991 Sequencing of human expressed sequence tags begins in Craig Venter's lab, an attempt to
capture the coding fraction of the human genome.
•1995 Craig Venter, Hamilton Smith, and colleagues at The Institute for Genomic Research (TIGR)
publish the first complete genome of a free-living organism, the bacterium Haemophilus influenzae.
The circular chromosome contains 1,830,137 bases and its publication in the journal Science marks
the first use of whole-genome shotgun sequencing, eliminating the need for initial mapping efforts.
•1996 Pål Nyrén and his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm
publish their method of pyrosequencing
•1998 Phil Green and Brent Ewing of the University of Washington publish "pyrex" for sequencer data
analysis.
•2000 Lynx Therapeutics publishes and markets "MPSS" - a parallelized, adapter/ligation-mediated,
bead-based sequencing technology, launching "next-generation" sequencing.
•2001 A draft sequence of the human genome is published.
•2004 454 Life Science markets a parallelized version of pyrosequencing. The first version of their
machine reduced sequencing costs 6-fold compared to automated Sanger sequencing, and was the second
of a new generation of sequencing technologies, after MPSS.

Platform
Library size
Data output
GS FLX
450bp Shot-gun sequencing
1 million reads
GS FLX
2 X 175bp paired-end library(3, 8, 20Kb insert)
1.2 million reads
Illumina HiSeq2000
50bp Single-end sequencing
280-300 million reads
Illumina HiSeq2000
100bp Single-end sequencing
280-300 million reads
Illumina HiSeq2000
2 X 50 pair-end sequencing
300 million reads
Illumina HiSeq2000
2 X 100 pair-end sequencing
300 million reads

•An overview of NGS platforms