How do the newest-latest DNA sequencing technologies work and what applications become $1000

possible with much cheaper sequencing?

Sequencing Reaction Miniaturization

Speed

Comparative Genome Sequencing

Efficiency
Sensitivity Throughput

Multiplexing Reactions
Microfluidics Software control

Integration of
Technologies
Nanoscaling
Robotics Data acquisition

Arun Ammayappan, Ernest Nyannor, Jason Sinclair, Senthilkumar Palaniyandi and Sandi Kirsch Automation Informatics

Introduction Current and Developing Techniques Flow diagram of SBS developed by 454 Life Sciences Pulsed Multi-line Excitation (PME)
Ligation Nebulization of
genome A method for multifluorescence
Early Sequencing Sequencing By Hybridization (SBH) Selection
(isolate
sstDNA library
with adaptors
discrimination of nucleotides
AB
avidin-biotin separated by CE.
Early sequencing was performed with tRNA through a The array contains all possible fragment
only) purification of
A/B fragment Four laser- four dye system
technique developed by Richard Holley, who published the first oligonucleotide sequences of a given
excites near absorption
structure of a tRNA in 1964. This involved breaking down RNA length.
maximum, uniformly intense
molecules, then puzzling the pieces back together. However, DNA of unknown sequence is incubated
emission signal.
this was extremely time consuming, and due to its large size, with the array.
Elimination of cross-talk between
such methods could not readily be used for DNA sequencing The target hybridizes to the array 4 bases (TACG)
cycled 42 times dye channels.
(Sanger 1988). Oliver wherever there is complementation to a Anneal sstDNA to Emulsify beads and PCR Clonal amplification Break microreactor
an excess of DNA reagents in water-in-oil occurs inside enrich for DNA Chemiluminescent
High fluorescent signal is
Frederick Sanger developed improved methods that allowed portion of the target. capture beads microreactors microreactors positive beads signal generation

Limitations Signal processing collected; it is easier to resolve

sequencing of some DNA up to 50 nucleotides in length. Hybridization of oligos are detected by to determine base
Difficult to reconstruct long Well diameter: 44m sequence and (Lewis, et al. 2005) the correct sequence.
However, he realized the potential of copying DNA instead of fluorescence. quality score
sequences. 200,000 reads obtained in
Less processing of fluorescent
degrading it (Sanger 1980). The probes are organized by overlaps parallel

Very large libraries are A single cloned amplified

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