Transcript Sequencing - Biochemistry at the University of

Introduction
Early Sequencing
• Early sequencing was performed with tRNA through a
technique developed by Richard Holley, who published the first
structure of a tRNA in 1964. This involved breaking down RNA
molecules, then puzzling the pieces back together. However,
this was extremely time consuming, and due to its large size,
such methods could not readily be used for DNA sequencing
(Sanger 1988).
• Frederick Sanger developed improved methods that allowed
sequencing of some DNA up to 50 nucleotides in length.
However, he realized the potential of copying DNA instead of
degrading it (Sanger 1980).
• In 1975 Sanger developed “the plus and minus method.” This
included the principal of chain termination during
polymerization, and was used to sequence an entire genome
(almost) – that of the φX 174 bacteriophage. Despite this
result, Sanger was not satisfied and kept searching for better
methodology (Sanger 1980).
• Sagner’s major breakthrough, which would become the basis
for subsequent techniques, came at a meeting in Germany
where Klaus Geider gave him a sample of ddTTP, which would
terminate chain polymerization upon incorporation. This would
be called “the di-deoxy method.”
dNTP
Addition of ddNTPs will terminate chain elongation. Location of ddNTP
insertion within a nucleotide chain can be determined using gel separation.
(image adapted from Sanger 1980)
• Sanger used this to finish the remainder of the φX 174 genome
in 1978.
These improvements and Maxim and Gilbert’s
chemical method of led to a large attraction towards sequencing
(Sanger 1988).
• Using the same methods as Sanger did in 1977, it would have
taken more than 1000,000 years to complete the human
genome. Three innovations came about that greatly expedited
the sequencing process: Shot-gun sequencing, PCR, and the
automation of sequencing. These developments led to the
publication of the first bacterial genome, H. influenzae Rd
KW20, in 1995 by Robert Fleischmann (Binnewies 2006). From
early sequencing of tRNA to the publication of a bacterial
genome took 30 years, but the groundwork was laid out for
future sequencing.
Human Genome Project
• Erwin Chargaff could not have imagined the rapid technological
advancement in computers and robotics when he made the
often quoted statement by critics of the human genome project;
“Even the smallest functional DNA varieties seen, those
occurring in certain small phages, must contain something like
5,000 nucleotides in a row. We may, therefore, leave the task of
reading the complete nucleotide sequence of a DNA to the 21st
century, which will, however, have other worries.”
• Progress was deemed too slow such that a decade to
publication of the 1st draft of the human genome, Bart Barrell
considered the HGP premature the outstanding advances made
then notwithstanding.
• The magnitude of progress made in rapid DNA sequencing has
been quite phenomenal.
• The prohibitive cost of sequencing a human genome can be
reduced through Novel detection assays, miniaturization in
instrumentation, microfluidic separation techniques, and
increase in number of assays per run.
• Novel detection assays are mostly modifications the Sanger
sequencing assay, but non-Sanger methods such as
pyrosequencing and several modification of it hold promise of
dramatically reducing the cost of genome sequencing.
Current and Developing Techniques
Sequencing By Hybridization (SBH)
• The array contains all possible
oligonucleotide sequences of a given
length.
• DNA of unknown sequence is incubated
with the array.
• The target hybridizes to the array
wherever there is complementation to a
Oliver
portion of the target.
Limitations
• Hybridization of oligos are detected by
•Difficult to reconstruct long
fluorescence.
sequences.
• The probes are organized by overlaps
•Very large libraries are
with one another to reconstruct the target
required.
sequence.
•The normal approach to SBH
is also sensitive to errors.
Latest Improvement and Advantages
• Universal bases are used instead of
normal oligonucleotides.
• By acting as spacers the universal
bases make consecutive probes less
dependent on one another.
• These are less sensitive to errors.
• Does not require larger libraries.
SBS involves detection of the identity of each nucleotide immediately
after its incorporation into a growing strand of DNA in a polymerase
reaction. The SBS includes "fluorescent in situ sequencing" (FISSEQ)
and the pyrosequencing method.
(Seo 2005)
• A different fluorophore is linked to each of the four bases through a
photocleavable linker.
• DNA polymerase incorporates complementary a single-nucleotide
analogue.
• Unique fluorescence emission detected depends upon the nt.
incorporated.
• Fluorophore is subsequently removed photochemically. The 3-OH
group is chemically regenerated and the cycle proceeds.
Advantages
• Allows parallel sequencing.
• Use of photons requires no additional chemical reagents.
• Clean products with no need of subsequent purification.
• avidin-biotin
purification of
A/B fragment
• 4 bases (TACG)
cycled 42 times
Emulsify beads and PCR
reagents in water-in-oil
microreactors
Clonal amplification
occurs inside
microreactors
Break microreactor
enrich for DNA
positive beads
• Chemiluminescent
signal generation
• Signal processing
to determine base
sequence and
quality score
• Well diameter: 44µm
• 200,000 reads obtained in
parallel
• A single cloned amplified
sstDNA bead is deposited
per well
• Non-Sanger nonfluorescence technique that
quantitatively measures released PPi
• Pyrogram corresponds to complementary base
Source: Biotage
Applications and advantages
• SNP analysis
• Ideal for rapidly mutating organisms
• Quantifications provide additional data
Limitations
• Short sequence reads
• Homopolymer repeat problems
Cyclic Reversible Terminator (CRT)
Sequencing by CRT consists of three steps; incorporation, imaging and
deprotection. The reversible terminator must be cleaved efficiently with
photocleaving groups like 2-nitrobenzyl group.
• Polony (polymerase colony) is amplified
product from single DNA molecule in
acrylamide gel.
• Sequencing done by the incorporation
of cleavable fluorescent labeled
nucleotide.
Advantage
• Scalability is easy by using 1μm
magnetic beads.
Disadvantage
• Failure in cleaving dye moiety.
Limitations
• Low readout length.
• Error prone.
Comparative Genome Sequencing
• Test DNA is hybridized with reference DNA to identify regions of genomic
differences.
• Genomic different regions are sequenced to identify SNPs.
Advantages
• Fast, accurate sequencing of the regions of interest.
“Lab-on-a-chip” concept: integration of all sequencing steps, including PCR
amplification, sample purification and capillary electrophoresis using the Sanger
sequencing method.
• Integration of Separation using 384
channels, accurately sequencing
560 bases with 99% accuracy.
• High throughput and decreased
(6x) analysis time.
• Reduced reagent and sample vol.
• Potential for low cost commercial
product.
Limitations
• Require longer read lengths.
• Rate limiting step: sample
preparation.
(Blazej, et al. 2006)
Applications
With quicker, faster, transportable, low-cost sequencing, applications include:
• Individual sequencing leading to personalized medicine- gene therapy.
• Rapid identification and characterization of pathogens.
• Profiling tumor subtypes for diagnosis and prognosis.
• Hypothesis testing for genotype/phenotype relationships.
• Understanding B- and T- cell receptor diversity to allow antibody selection.
Ethical Issues
Advances and declining costs for sequencing technology will yield
accessible genotype- phenotypic information to the scientific community. Rising
issues within the scientific community and the public include identifiably of
individuals, intellectual property vs. individual property rights to genomic
sequences, requirements to “share” research results, and targeting research
towards/away from certain races and cultures based on cost benefits.
Conclusion
(Mitra.)
• Determines the order of nonconsecutive nucleotide additions.
• Cy3-labeled-UTP is incorporated into the primer strand, donor dye. Subsequent
incorporation of a complementary Cy5-labeled-UTP or Cy5-labeled-dCTP
substrate results ins a spFRET signal.
• Photobleaching of Cy5 dye addition of natural nucleotides dATP and dGTP
addition of Cy5-labeled dNTP.
Advantages
• High degree of parallelization.
• Sparing use of reagents.
Sequencing Reaction Miniaturization
Informatics
Multiplexing Reactions
Integration of
Technologies
Automation
Data acquisition
Microfluidic Separation Platforms
• sstDNA library
with adaptors
Single-Pair FRET (spFRET)
Cycle continues
Robotics
Software control
Pyrosequencing
Selection
(isolate
AB
fragment
only)
Polony Technology
Sequencing-By-Synthesis (SBS)
Microfluidics
Throughput
Single-nucleotide addition (SNA)
• Nebulization of
genome
Advantages
• Avoids gel electrophoresis, functions in highly parallel fashion, high throughput,
speed and accuracy.
(Harvard Nanopore Group)
Sensitivity
• A method for multifluorescence
discrimination of nucleotides
separated by CE.
• Four laser- four dye system
excites near absorption
maximum, uniformly intense
emission signal.
• Elimination of cross-talk between
dye channels.
• High fluorescent signal is
collected; it is easier to resolve
(Lewis, et al. 2005)
the correct sequence.
• Less processing of fluorescent
data is required.
Future Implications
• Potential for a transportable and compact DNA sequencing system.
• Higher sensitivity, quicker analysis, and lower cost: shortened preparation
time, reduced sample and reagent volumes, and less data processing.
Ligation
Nanopore Sequencing
• Utilizes a nanoscale device that translocates polymer molecules in
sequential monomer order through a very small volume of space.
• Includes a detector that directly converts characteristic features of the
translocating polymer into an electrical signal. Transduction and
recognition occur in real time, on a molecule-by-molecule basis. It can
probe thousands of different molecules in a few minutes.
• It can probe very long lengths of DNA.
Efficiency
Pulsed Multi-line Excitation (PME)
Flow diagram of SBS developed by 454 Life Sciences
Anneal sstDNA to
an excess of DNA
capture beads
$1000
Speed
Nanoscaling
Arun Ammayappan, Ernest Nyannor, Jason Sinclair, Senthilkumar Palaniyandi and Sandi Kirsch
Comparative Genome Sequencing
How do the newest-latest DNA sequencing technologies work and what applications become
possible with much cheaper sequencing?
With the advancements in sequencing and its marriage with computer
science, the face of biology has been altered. Biology will merge with computer
science, mathematics, and physics as never before (Yao 2002), adding the
advancements made. However, lets not forget about a meeting in Germany 40
years ago when one man told another that he had some ddTTP.
References
• Bart Barrell, 1991. DNA sequencing: present limitation and prospects for the future. FASEB J: 5: 40-45
• Robert G. Blazej et al., 2006. Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA
sequencing. PNAS: 103(19): 7240-7245
• Biotage. http://www.pyrosequencing.com/DynPage.aspx?id=8726&mn1=1366
• Morris W. Foster and Richard R. Sharp, 2006. Ethical issues in medical-sequencing research: implications of
genotype-phenotype studies for individuals and populations. Hum. Mol. Gen.: 15(R1): R45-R49
• E.K.Lewis,et al., 2005. Color-blind fluorescence detection for four-color DNA sequencing. PNAS:102(15):5346-51
• Oliver. http://www.chem.brown.edu/faculty/oliver/slide1.htm
• Mitra. http://cbcg.lbl.gov/Genome9/Talks/mitra.pdf
• M. L. Metzker, 2005. Emerging technologies in DNA sequencing. Genome Res.:15(12):1767-76
• NimbleGen. http://www.nimblegen.com/products/cgr/index.html
• Tae Seok Seo et al., 2005. Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent
nucleotides. PNAS: 102(17): 5926-5931
• 454 Life Sciences. http://www.454.com/enabling-technology/the-process.asp
• Caitlin Smith. 2005. Genomics: Getting down to details. Nature 435, 991-994
• Harvard Nanopore Group. http://www.mcb.harvard.edu/branton/projects-NanoporeSequencing.htm
• Tim T. Binnewies et al., 2006. Ten years of bacterial genome sequencing: comparative-genomics-based
discoveries. Func. Integ. Genomics 6:165-85
• Frederick Sagner, 1980. Determination of Nucleotide Sequences in DNA. Nobel Lectures, Chemistry 1971-80
• Frederick Sanger. Sequences, Sequences, Sequences. Annu. Rev. Biochem. 57:1-28, 1988
• Toru Yao, 2002. Bioinformatics for the genomic sciences and towards systems biology. Progress in Biophysics
and Molecular Biology 80:23-42