Transmission electron microscopy DNA sequencing
Encyclopedia
Transmission electron microscopy DNA sequencing is an emerging third-generation, single-molecule sequencing
technology that uses transmission electron microscopy techniques
. DNA
is visible under the electron microscope
; however, it must be labeled with heavy atoms so that the DNA bases
can be clearly visualized. In addition, specialized imaging techniques and aberration corrected optics are beneficial for obtaining the resolution required to image the labeled DNA molecule. Transmission electron microscopy DNA sequencing advantageously may provide extremely long read lengths, but it is not yet commercially available.
and Francis Crick
deduced the structure of DNA, and nearly two decades before Frederick Sanger
published the first method for rapid DNA sequencing
, Richard Feynman
, an American physicist, envisioned the electron microscope
as the tool that would one day allow biologists to “see the order of bases
in the DNA
chain”. Feynman believed that if the electron microscope could be made powerful enough, then it would become possible to visualize the atomic structure of any and all chemical compounds, including DNA.
To this day, despite the invention of a multitude of chemical and fluorescent sequencing technologies, microscopy
is still being explored as a means of performing single-molecule DNA sequencing. Two biotechnology companies have conceived of methods for high throughput, direct detection of DNA bases by transmission electron microscopy
; however, these studies are still in their infancy and are far from being commercially available. The following progress in these technologies has been reported:
has the capacity to obtain a resolution of up to 100 pm, whereby microscopic biomolecules and structures such as viruses, ribosomes, proteins, lipids, small molecules and even single atoms can be observed.
Although DNA
is visible when observed with the electron microscope, the resolution of the image obtained is not high enough to allow for deciphering the sequence of the individual bases
, i.e., DNA sequencing
. However, upon differential labeling of the DNA bases with heavy atoms or metals, it is possible to both visualize and distinguish between the individual bases. Therefore, electron microscopy in conjunction with differential heavy atom DNA labeling could be used to directly image the DNA in order to determine its sequence.
As in a standard polymerase chain reaction (PCR)
, the double stranded DNA molecules to be sequenced must be denatured
before the second strand can be synthesized with labeled nucleotides.
The elements that make up biological molecules (C
, H
, N
, O
, P
, S
) are too light (low atomic number, Z
) to be clearly visualized as individual atoms by transmission electron microscopy
. To circumvent this problem, the DNA bases
can be labeled with heavier atoms (higher Z). Each nucleotide
is tagged with a characteristic heavy label, so that they can be distinguished in the transmission electron micrograph.
The DNA molecules must be stretched out on a thin, solid substrate so that order of the labeled bases will be clearly visible on the electron micrograph. Molecular combing
is a technique that utilizes the force of a receding air-water interface to extend DNA molecules, leaving them irreversibly bound to a silane layer once dry. This is one means by which alignment of the DNA on a solid substrate may be achieved.
Transmission electron microscopy (TEM) produces high magnification, high resolution images by passing a beam of electrons through a very thin sample. Whereas atomic resolution has been demonstrated with conventional TEM, further improvement in spatial resolution requires correcting the spherical
and chromatic aberrations of the microscope lenses
. This has only been possible in scanning transmission electron microscopy
where the image is obtained by scanning the object with a finely focused electron beam, in a way similar to a cathode ray tube
. However, the achieved improvement in resolution comes together with irradiation of the studied object by much higher beam intensities, the concomitant sample damage and the associated imaging artefacts. Different imaging techniques are applied depending on whether the sample contains heavy or light atoms:
Dark and bright spots on the electron micrograph, corresponding to the differentially labeled DNA bases, are analyzed by computer software.
When sequencing a genome, it must be broken down into pieces that are short enough to be sequenced in a single read. These reads must then be put back together like a jigsaw puzzle by aligning the regions that overlap between reads; this process is called de novo genome assembly. The longer the read length that a sequencing platform provides, the longer the overlapping regions, and the easier it is to assemble the genome. From a computational perspective, microfluidic Sanger sequencing
is still the most effective way to sequence and assemble genomes for which no reference genome
sequence exists. The relatively long read lengths provide substantial overlap between individual sequencing reads, which allows for greater statistical confidence in the assembly. In addition, long Sanger reads are able to span most regions of repetitive DNA sequence
which otherwise confound sequence assembly by causing false alignments. However, de novo genome assembly by Sanger sequencing is extremely expensive and time consuming. Second generation sequencing technologies, while less expensive, are generally unfit for de novo genome assembly due to short read lengths. In general, third generation sequencing technologies, including transmission electron microscopy DNA sequencing, aim to improve read length while maintaining low sequencing cost. Thus, as third generation sequencing technologies improve, rapid and inexpensive de novo genome assembly will become a reality.
A haplotype
is a series of linked allele
s that are inherited together on a single chromosome. DNA sequencing can be used to genotype
all of the single nucleotide polymorphisms (SNPs) that constitute a haplotype. However, short DNA sequencing reads often cannot be phased; that is, heterozygous variants cannot be confidently assigned to the correct haplotype. In fact, haplotyping with short read DNA sequencing data requires very high coverage (average >50x coverage of each DNA base) to accurately identify SNPs, as well as additional sequence data from the parents so that Mendelian transmission
can be used to estimate the haplotypes. Sequencing technologies that generate long reads, including transmission electron microscopy DNA sequencing, can capture entire haploblocks in a single read. That is, haplotypes are not broken up among multiple reads, and the genetically linked alleles remain together in the sequencing data. Therefore, long reads make haplotyping easier and more accurate, which is beneficial to the field of population genetics
.
Genes are normally present in two copies in the diploid human genome; genes that deviate from this standard copy number are referred to as copy number variants (CNVs). Copy number variation can be benign (these are usually common variants, called copy number polymorphisms) or pathogenic. CNVs are detected by fluorescence in situ hybridization (FISH) or comparative genomic hybridization (CGH)
. To detect the specific breakpoints at which a deletion occurs, or to detect genomic lesions introduced by a duplication or amplification event, CGH can be performed using a tiling array
(array CGH
), or the variant region can be sequenced. Long sequencing reads are especially useful for analyzing duplications or amplifications, as it is possible to analyze the orientation of the amplified segments if they are captured in a single sequencing read.
Cancer genomics, or oncogenomics
, is an emerging field in which high-throughput, second generation DNA sequencing technology is being applied to sequence entire cancer genomes. Analyzing this short read sequencing data encompasses all of the problems associated with de novo genome assembly using short read data. Furthermore, cancer genomes are often aneuploid
. These aberrations, which are essentially large scale copy number variants, can be analyzed by second-generation sequencing technologies using read frequency to estimate the copy number. Longer reads would, however, provide a more accurate picture of copy number, orientation of amplified regions, and SNPs present in cancer genomes.
The microbiome
refers the total collection of microbes present in a microenvironment and their respective genomes. For example, an estimated 100 trillion microbial cells colonize the human body at any given time. The human microbiome is of particular interest, as these commensal bacteria
are important for human health and immunity. Most of the Earth's bacterial genomes have not yet been sequenced; undertaking a microbiome sequencing project would require extensive de novo genome assembly, a prospect which is daunting with short read DNA sequencing technologies. Longer reads would greatly facilitate the assembly of new microbial genomes.
Many non-Sanger second- and third-generation DNA sequencing technologies have been or are currently being developed with the common aim of increasing throughput and decreasing cost such that personalized genetic medicine can be fully realized.
Both the US$10 million Archon X Prize
for Genomics supported by the X Prize Foundation
(Santa Monica, CA, USA) and the US$70 million in grant awards supported by the National Human Genome Research Institute
of the National Institutes of Health
(NIH-NHGRI) are fueling the rapid burst of research activity in the development of new DNA sequencing technologies.
Since different approaches, techniques, and strategies are what define each DNA sequencing technology, each has its own strengths and weaknesses. Comparison of important parameters between various second- and third-generation DNA sequencing technologies are presented in Table 1.
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
technology that uses transmission electron microscopy techniques
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
. DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
is visible under the electron microscope
Electron microscope
An electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than...
; however, it must be labeled with heavy atoms so that the DNA bases
Nucleobase
Nucleobases are a group of nitrogen-based molecules that are required to form nucleotides, the basic building blocks of DNA and RNA. Nucleobases provide the molecular structure necessary for the hydrogen bonding of complementary DNA and RNA strands, and are key components in the formation of stable...
can be clearly visualized. In addition, specialized imaging techniques and aberration corrected optics are beneficial for obtaining the resolution required to image the labeled DNA molecule. Transmission electron microscopy DNA sequencing advantageously may provide extremely long read lengths, but it is not yet commercially available.
History
Only a few years after James WatsonJames D. Watson
James Dewey Watson is an American molecular biologist, geneticist, and zoologist, best known as one of the co-discoverers of the structure of DNA in 1953 with Francis Crick...
and Francis Crick
Francis Crick
Francis Harry Compton Crick OM FRS was an English molecular biologist, biophysicist, and neuroscientist, and most noted for being one of two co-discoverers of the structure of the DNA molecule in 1953, together with James D. Watson...
deduced the structure of DNA, and nearly two decades before Frederick Sanger
Frederick Sanger
Frederick Sanger, OM, CH, CBE, FRS is an English biochemist and a two-time Nobel laureate in chemistry, the only person to have been so. In 1958 he was awarded a Nobel prize in chemistry "for his work on the structure of proteins, especially that of insulin"...
published the first method for rapid DNA sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
, Richard Feynman
Richard Feynman
Richard Phillips Feynman was an American physicist known for his work in the path integral formulation of quantum mechanics, the theory of quantum electrodynamics and the physics of the superfluidity of supercooled liquid helium, as well as in particle physics...
, an American physicist, envisioned the electron microscope
Electron microscope
An electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than...
as the tool that would one day allow biologists to “see the order of bases
Nucleobase
Nucleobases are a group of nitrogen-based molecules that are required to form nucleotides, the basic building blocks of DNA and RNA. Nucleobases provide the molecular structure necessary for the hydrogen bonding of complementary DNA and RNA strands, and are key components in the formation of stable...
in the DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
chain”. Feynman believed that if the electron microscope could be made powerful enough, then it would become possible to visualize the atomic structure of any and all chemical compounds, including DNA.
To this day, despite the invention of a multitude of chemical and fluorescent sequencing technologies, microscopy
Microscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
is still being explored as a means of performing single-molecule DNA sequencing. Two biotechnology companies have conceived of methods for high throughput, direct detection of DNA bases by transmission electron microscopy
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
; however, these studies are still in their infancy and are far from being commercially available. The following progress in these technologies has been reported:
- 1970 Albert Crewe developed the high-angle annular dark-field imagingAnnular dark-field imagingAnnular dark-field imaging is a method of mapping samples in a scanning transmission electron microscope . These images are formed by collecting scattered electrons with an annular dark-field detector....
(HAADF) imaging technique in a scanning transmission electron microscope. Using this technique, he visualized individual heavy atoms on thin amorphous carbon films. - April 2008: ZS Genetics presented its plans for development of a transmission electron microscopy-based single-molecule sequencing platform at the Cambridge Health-tech Institute (CHI) Sequencing Conference in San Diego, held from 23–24 April 2008.
- March 2010: Krivanek and colleagues reported several technical improvements to the HAADF method, including a combination of aberration corrected electron optics and low accelerating voltage. The latter is crucial for imaging biological objects, as it allows to reduce damage by the beam and increase the image contrast for light atoms. As a result, single atom substitutions in a boron nitride monolayer could be imaged. Halcyon Molecular is developing its single-molecule sequencing platform based on the technology utilized in this paper.
- September 2010: The Toste research group at University of California, BerkeleyUniversity of California, BerkeleyThe University of California, Berkeley , is a teaching and research university established in 1868 and located in Berkeley, California, USA...
, received an Advanced Sequencing Technology Award from the National Human Genome Research InstituteNational Human Genome Research InstituteThe National Human Genome Research Institute is a division of the National Institutes of Health, located in Bethesda, Maryland.NHGRI began as the National Center for Human Genome Research , which was established in 1989 to carry out the role of the NIH in the International Human Genome Project...
to continue research into single-molecule sequencing by transmission electron microscopy, in collaboration with Halcyon Molecular.
Principle
The electron microscopeElectron microscope
An electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than...
has the capacity to obtain a resolution of up to 100 pm, whereby microscopic biomolecules and structures such as viruses, ribosomes, proteins, lipids, small molecules and even single atoms can be observed.
Although DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
is visible when observed with the electron microscope, the resolution of the image obtained is not high enough to allow for deciphering the sequence of the individual bases
Nucleobase
Nucleobases are a group of nitrogen-based molecules that are required to form nucleotides, the basic building blocks of DNA and RNA. Nucleobases provide the molecular structure necessary for the hydrogen bonding of complementary DNA and RNA strands, and are key components in the formation of stable...
, i.e., DNA sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
. However, upon differential labeling of the DNA bases with heavy atoms or metals, it is possible to both visualize and distinguish between the individual bases. Therefore, electron microscopy in conjunction with differential heavy atom DNA labeling could be used to directly image the DNA in order to determine its sequence.
Workflow
Step 1 – DNA denaturation
As in a standard polymerase chain reaction (PCR)
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
, the double stranded DNA molecules to be sequenced must be denatured
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
before the second strand can be synthesized with labeled nucleotides.
Step 2 – Heavy atom labeling
The elements that make up biological molecules (C
Carbon
Carbon is the chemical element with symbol C and atomic number 6. As a member of group 14 on the periodic table, it is nonmetallic and tetravalent—making four electrons available to form covalent chemical bonds...
, H
Hydrogen
Hydrogen is the chemical element with atomic number 1. It is represented by the symbol H. With an average atomic weight of , hydrogen is the lightest and most abundant chemical element, constituting roughly 75% of the Universe's chemical elemental mass. Stars in the main sequence are mainly...
, N
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
, O
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
, P
Phosphorus
Phosphorus is the chemical element that has the symbol P and atomic number 15. A multivalent nonmetal of the nitrogen group, phosphorus as a mineral is almost always present in its maximally oxidized state, as inorganic phosphate rocks...
, S
Sulfur
Sulfur or sulphur is the chemical element with atomic number 16. In the periodic table it is represented by the symbol S. It is an abundant, multivalent non-metal. Under normal conditions, sulfur atoms form cyclic octatomic molecules with chemical formula S8. Elemental sulfur is a bright yellow...
) are too light (low atomic number, Z
Atomic number
In chemistry and physics, the atomic number is the number of protons found in the nucleus of an atom and therefore identical to the charge number of the nucleus. It is conventionally represented by the symbol Z. The atomic number uniquely identifies a chemical element...
) to be clearly visualized as individual atoms by transmission electron microscopy
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
. To circumvent this problem, the DNA bases
Nucleobase
Nucleobases are a group of nitrogen-based molecules that are required to form nucleotides, the basic building blocks of DNA and RNA. Nucleobases provide the molecular structure necessary for the hydrogen bonding of complementary DNA and RNA strands, and are key components in the formation of stable...
can be labeled with heavier atoms (higher Z). Each nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
is tagged with a characteristic heavy label, so that they can be distinguished in the transmission electron micrograph.
- ZS Genetics proposes using three heavy labels: bromineBromineBromine ") is a chemical element with the symbol Br, an atomic number of 35, and an atomic mass of 79.904. It is in the halogen element group. The element was isolated independently by two chemists, Carl Jacob Löwig and Antoine Jerome Balard, in 1825–1826...
(Z=35), iodineIodineIodine is a chemical element with the symbol I and atomic number 53. The name is pronounced , , or . The name is from the , meaning violet or purple, due to the color of elemental iodine vapor....
(Z=53), and trichloromethane (total Z=63). These would appear as differential dark and light spots on the micrograph, and the fourth DNA base would remain unlabeled. - Halcyon Molecular, in collaboration with the Toste group, proposes that purinePurineA purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....
and pyrimidinePyrimidinePyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
bases can be functionalized with platinum diamine or osmium tetraoxide bipyridine, respectively. Heavy metal atoms such as osmiumOsmiumOsmium is a chemical element with the symbol Os and atomic number 76. Osmium is a hard, brittle, blue-gray or blue-blacktransition metal in the platinum family, and is the densest natural element. Osmium is twice as dense as lead. The density of osmium is , slightly greater than that of iridium,...
(Z=76), iridiumIridiumIridium is the chemical element with atomic number 77, and is represented by the symbol Ir. A very hard, brittle, silvery-white transition metal of the platinum family, iridium is the second-densest element and is the most corrosion-resistant metal, even at temperatures as high as 2000 °C...
(Z=77), goldGoldGold is a chemical element with the symbol Au and an atomic number of 79. Gold is a dense, soft, shiny, malleable and ductile metal. Pure gold has a bright yellow color and luster traditionally considered attractive, which it maintains without oxidizing in air or water. Chemically, gold is a...
(Z=79), or uraniumUraniumUranium is a silvery-white metallic chemical element in the actinide series of the periodic table, with atomic number 92. It is assigned the chemical symbol U. A uranium atom has 92 protons and 92 electrons, of which 6 are valence electrons...
(Z=92) can then form metal-metal bonds with these functional groups to label the individual bases.
Step 3 – DNA alignment on substrate
The DNA molecules must be stretched out on a thin, solid substrate so that order of the labeled bases will be clearly visible on the electron micrograph. Molecular combing
Chromosome combing
Chromosome combing is a technique used to produce an array of uniformly stretched DNA that is then highly suitable for nucleic acid hybridization studies such as fluorescent in situ hybridisation which benefit from the uniformity of stretching, the easy access to the hybridisation target...
is a technique that utilizes the force of a receding air-water interface to extend DNA molecules, leaving them irreversibly bound to a silane layer once dry. This is one means by which alignment of the DNA on a solid substrate may be achieved.
Step 4 – TEM imaging
Spherical aberration
thumb|right|Spherical aberration. A perfect lens focuses all incoming rays to a point on the [[Optical axis|optic axis]]. A real lens with spherical surfaces suffers from spherical aberration: it focuses rays more tightly if they enter it far from the optic axis than if they enter closer to the...
and chromatic aberrations of the microscope lenses
Objective (optics)
In an optical instrument, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be single lenses or mirrors, or combinations of several optical elements. They are used in microscopes, telescopes,...
. This has only been possible in scanning transmission electron microscopy
Scanning transmission electron microscopy
A scanning transmission electron microscope is a type of transmission electron microscope . As with any transmission illumination scheme, the electrons pass through a sufficiently thin specimen...
where the image is obtained by scanning the object with a finely focused electron beam, in a way similar to a cathode ray tube
Cathode ray tube
The cathode ray tube is a vacuum tube containing an electron gun and a fluorescent screen used to view images. It has a means to accelerate and deflect the electron beam onto the fluorescent screen to create the images. The image may represent electrical waveforms , pictures , radar targets and...
. However, the achieved improvement in resolution comes together with irradiation of the studied object by much higher beam intensities, the concomitant sample damage and the associated imaging artefacts. Different imaging techniques are applied depending on whether the sample contains heavy or light atoms:
- Annular dark-field imagingAnnular dark-field imagingAnnular dark-field imaging is a method of mapping samples in a scanning transmission electron microscope . These images are formed by collecting scattered electrons with an annular dark-field detector....
measures the scattering of electrons as they deflect off the nuclei of the atoms in the transmission electron microscopy sample. This is best suited to samples containing heavy atoms, as they cause more scattering of electrons. The technique has been used to image atoms as light as boronBoronBoron is the chemical element with atomic number 5 and the chemical symbol B. Boron is a metalloid. Because boron is not produced by stellar nucleosynthesis, it is a low-abundance element in both the solar system and the Earth's crust. However, boron is concentrated on Earth by the...
, nitrogenNitrogenNitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
, and carbonCarbonCarbon is the chemical element with symbol C and atomic number 6. As a member of group 14 on the periodic table, it is nonmetallic and tetravalent—making four electrons available to form covalent chemical bonds...
; however, the signal is very weak for such light atoms. If annular dark-field microscopy is put to use for transmission electron microscopy DNA sequencing, it will certainly be necessary to label the DNA bases with heavy atoms so that a strong signal can be detected. - Annular bright-field imaging detects electrons transmitted directly through the sample, and measures the wave interference produced by their interactions with the atomic nuclei. This technique can detect light atoms with greater sensitivity than annular dark-field imaging methods. In fact, oxygenOxygenOxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
, nitrogenNitrogenNitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
, lithiumLithiumLithium is a soft, silver-white metal that belongs to the alkali metal group of chemical elements. It is represented by the symbol Li, and it has the atomic number 3. Under standard conditions it is the lightest metal and the least dense solid element. Like all alkali metals, lithium is highly...
, and hydrogenHydrogenHydrogen is the chemical element with atomic number 1. It is represented by the symbol H. With an average atomic weight of , hydrogen is the lightest and most abundant chemical element, constituting roughly 75% of the Universe's chemical elemental mass. Stars in the main sequence are mainly...
in crystalline solids have been imaged using annular bright-field electron microscopy. Thus, it is theoretically possible to obtain direct images of the atoms in the DNA chain; however, the structure of DNA is much less geometric than crystalline solids, so direct imaging without prior labeling may not be achievable.
Step 5 – Data analysis
Dark and bright spots on the electron micrograph, corresponding to the differentially labeled DNA bases, are analyzed by computer software.
Applications
Transmission electron microscopy DNA sequencing is not yet commercially available, but the long read lengths that this technology may one day provide will make it useful in a variety of contexts.De novo genome assembly
When sequencing a genome, it must be broken down into pieces that are short enough to be sequenced in a single read. These reads must then be put back together like a jigsaw puzzle by aligning the regions that overlap between reads; this process is called de novo genome assembly. The longer the read length that a sequencing platform provides, the longer the overlapping regions, and the easier it is to assemble the genome. From a computational perspective, microfluidic Sanger sequencing
Microfluidic Sanger Sequencing
The completion of the Human Genome Project has been a cornerstone in the advancement of biological studies. The outcomes of obtaining a complete reference map of the human genome have ushered in the post-genome era of studies...
is still the most effective way to sequence and assemble genomes for which no reference genome
Reference genome
A reference genome is a digital nucleic acid sequence database, assembled by scientists as a representative example of a species' genetic code. As they are often assembled from the sequencing of DNA from a number of donors, reference genomes do not accurately represent the genetic code of any...
sequence exists. The relatively long read lengths provide substantial overlap between individual sequencing reads, which allows for greater statistical confidence in the assembly. In addition, long Sanger reads are able to span most regions of repetitive DNA sequence
Repeated sequence (DNA)
In the study of DNA sequences, one can distinguish two main types of repeated sequence:*Tandem repeats:**Satellite DNA**Minisatellite**Microsatellite*Interspersed repeats:**SINEs...
which otherwise confound sequence assembly by causing false alignments. However, de novo genome assembly by Sanger sequencing is extremely expensive and time consuming. Second generation sequencing technologies, while less expensive, are generally unfit for de novo genome assembly due to short read lengths. In general, third generation sequencing technologies, including transmission electron microscopy DNA sequencing, aim to improve read length while maintaining low sequencing cost. Thus, as third generation sequencing technologies improve, rapid and inexpensive de novo genome assembly will become a reality.
Full haplotypes
A haplotype
Haplotype
A haplotype in genetics is a combination of alleles at adjacent locations on the chromosome that are transmitted together...
is a series of linked allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
s that are inherited together on a single chromosome. DNA sequencing can be used to genotype
Genotyping
Genotyping is the process of determining differences in the genetic make-up of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their...
all of the single nucleotide polymorphisms (SNPs) that constitute a haplotype. However, short DNA sequencing reads often cannot be phased; that is, heterozygous variants cannot be confidently assigned to the correct haplotype. In fact, haplotyping with short read DNA sequencing data requires very high coverage (average >50x coverage of each DNA base) to accurately identify SNPs, as well as additional sequence data from the parents so that Mendelian transmission
Mendelian inheritance
Mendelian inheritance is a scientific description of how hereditary characteristics are passed from parent organisms to their offspring; it underlies much of genetics...
can be used to estimate the haplotypes. Sequencing technologies that generate long reads, including transmission electron microscopy DNA sequencing, can capture entire haploblocks in a single read. That is, haplotypes are not broken up among multiple reads, and the genetically linked alleles remain together in the sequencing data. Therefore, long reads make haplotyping easier and more accurate, which is beneficial to the field of population genetics
Population genetics
Population genetics is the study of allele frequency distribution and change under the influence of the four main evolutionary processes: natural selection, genetic drift, mutation and gene flow. It also takes into account the factors of recombination, population subdivision and population...
.
Copy number variants
Genes are normally present in two copies in the diploid human genome; genes that deviate from this standard copy number are referred to as copy number variants (CNVs). Copy number variation can be benign (these are usually common variants, called copy number polymorphisms) or pathogenic. CNVs are detected by fluorescence in situ hybridization (FISH) or comparative genomic hybridization (CGH)
Comparative genomic hybridization
Comparative genomic hybridization or Chromosomal Microarray Analysis is a molecular-cytogenetic method for the analysis of copy number changes in the DNA content of a given subject's DNA and often in tumor cells....
. To detect the specific breakpoints at which a deletion occurs, or to detect genomic lesions introduced by a duplication or amplification event, CGH can be performed using a tiling array
Tiling array
Tiling Arrays are a subtype of microarray chips. Like traditional microarrays, they function by hybridizing labeled DNA or RNA target molecules to probes fixed onto a solid surface. Tiling arrays differ from traditional microarrays in the nature of the probes...
(array CGH
Array comparative genomic hybridization
Array-comparative genomic hybridization is a technique to detect genomic copy number variations at a higher resolution level than chromosome-based comparative genomic hybridization .-Process:DNA from...
), or the variant region can be sequenced. Long sequencing reads are especially useful for analyzing duplications or amplifications, as it is possible to analyze the orientation of the amplified segments if they are captured in a single sequencing read.
Cancer
Cancer genomics, or oncogenomics
Oncogenomics
Oncogenomics is relatively new sub-field of genomics, which applies high throughput technologies to characterize genes associated with cancer. Oncogenomics is synonymous with "cancer genomics". Cancer is a genetic disease caused by accumulation of mutations to DNA leading to unrestrained cell...
, is an emerging field in which high-throughput, second generation DNA sequencing technology is being applied to sequence entire cancer genomes. Analyzing this short read sequencing data encompasses all of the problems associated with de novo genome assembly using short read data. Furthermore, cancer genomes are often aneuploid
Aneuploidy
Aneuploidy is an abnormal number of chromosomes, and is a type of chromosome abnormality. An extra or missing chromosome is a common cause of genetic disorders . Some cancer cells also have abnormal numbers of chromosomes. Aneuploidy occurs during cell division when the chromosomes do not separate...
. These aberrations, which are essentially large scale copy number variants, can be analyzed by second-generation sequencing technologies using read frequency to estimate the copy number. Longer reads would, however, provide a more accurate picture of copy number, orientation of amplified regions, and SNPs present in cancer genomes.
Microbiome sequencing
The microbiome
Microbiome
A microbiome is the totality of microbes, their genetic elements , and environmental interactions in a defined environment. A defined environment could, for example, be the gut of a human being or a soil sample. Thus, microbiome usually includes microbiota and their complete genetic elements...
refers the total collection of microbes present in a microenvironment and their respective genomes. For example, an estimated 100 trillion microbial cells colonize the human body at any given time. The human microbiome is of particular interest, as these commensal bacteria
Commensalism
In ecology, commensalism is a class of relationship between two organisms where one organism benefits but the other is neutral...
are important for human health and immunity. Most of the Earth's bacterial genomes have not yet been sequenced; undertaking a microbiome sequencing project would require extensive de novo genome assembly, a prospect which is daunting with short read DNA sequencing technologies. Longer reads would greatly facilitate the assembly of new microbial genomes.
Strengths and weaknesses
Compared to other second- and third-generation DNA sequencing technologies, transmission electron microscopy DNA sequencing has a number of potential key strengths and weaknesses, which will ultimately determine its usefulness and prominence as a future DNA sequencing technology.Strengths
- Longer read lengths: ZS Genetics has estimated potential read lengths of transmission electron microscopy DNA sequencing to be 10,000 to 20,000 base pairs with a rate of 1.7 billion base pairs per day. Such long read lengths would allow easier de novo genome assembly and direct detection of haplotypes, among other applications.
- Lower cost: Transmission electron microscopy DNA sequencing is estimated to cost just US$5,000-US$10,000 per human genome, compared to the more expensive second-generation DNA sequencing alternatives.
- No dephasing: Dephasing of the DNA strands due to loss in synchronicity during synthesis is a major problem of second-generation sequencing technologies. For transmission electron microscopy DNA sequencing and several other third-generation sequencing technologies, sychronization of the reads is unnecessary as only one molecule is being read at a time.
- Shorter turnaround time: The capacity to read native fragments of DNA renders complex template preparation an unnecessary step in the general workflow of whole genome sequencing. Consequently, shorter turnaround times are possible.
Weaknesses
- High capital cost: A transmission electron microscope with sufficient resolution required for transmission electron microscopy DNA sequencing costs approximately US$1,000,000, therefore pursuing DNA sequencing by this method requires a substantial investment.
- Technically challenging: Selective heavy atom labeling and attaching and straightening the labeled DNA to a substrate are a serious technical challenge. Further, the DNA sample should be stable to the high vacuum of electron microscope and irradiation by a focused beam of high-energy electrons.
- Potential PCR bias and artefacts: Although PCR is only being utilized in transmission electron microscopy DNA sequencing as a means to label the DNA strand with heavy atoms or metals, there could be the possibility of introducing bias in template representation or errors during the single amplification.
Comparison to other sequencing technologies
Many non-Sanger second- and third-generation DNA sequencing technologies have been or are currently being developed with the common aim of increasing throughput and decreasing cost such that personalized genetic medicine can be fully realized.
Both the US$10 million Archon X Prize
Archon X Prize
The Archon X Prize for Genomics, the second X Prize to be offered by the X Prize Foundation, based in Santa Monica, California, was announced on October 4, 2006. The Archon X Prize in genomics is a joint effort of the X Prize Foundation and the J...
for Genomics supported by the X Prize Foundation
X Prize Foundation
The X PRIZE Foundation is a non-profit organization that designs and manages public competitions intended to encourage technological development that could benefit mankind....
(Santa Monica, CA, USA) and the US$70 million in grant awards supported by the National Human Genome Research Institute
National Human Genome Research Institute
The National Human Genome Research Institute is a division of the National Institutes of Health, located in Bethesda, Maryland.NHGRI began as the National Center for Human Genome Research , which was established in 1989 to carry out the role of the NIH in the International Human Genome Project...
of the National Institutes of Health
National Institutes of Health
The National Institutes of Health are an agency of the United States Department of Health and Human Services and are the primary agency of the United States government responsible for biomedical and health-related research. Its science and engineering counterpart is the National Science Foundation...
(NIH-NHGRI) are fueling the rapid burst of research activity in the development of new DNA sequencing technologies.
Since different approaches, techniques, and strategies are what define each DNA sequencing technology, each has its own strengths and weaknesses. Comparison of important parameters between various second- and third-generation DNA sequencing technologies are presented in Table 1.
| Platform | Generation | Read length (bp) | Accuracy | Cost per human genome (US$) | Cost of instrument (US$) | Run time (h/Gbp) |
|---|---|---|---|---|---|---|
| Massively parallel pyrosequencing by synthesis (Roche/454: GS FLX Titanium Series) | Second | 400–500 | Q20 read length of 40 bases (99% at 400 bases and higher for prior bases) | 1,000,000 | 500,000 | 75 |
| Sequencing by synthesis (Illumina/Solexa: Genome Analyzer IIx) | Second | 2×75 | Base call with Q30 (>70%) | 60,000 | 450,000 | 56 |
| Bead-based massively parallel clonal ligation based sequencing (Applied Biosystems: SOLiD 3 System) | Second | 100 | 99.94% | 60,000 | 591,000 | 42 |
| Massively parallel single-molecule sequencing by synthesis (Helicos/Stanford Univ.) | Third | 30–35 | 99.995% at >20×coverage (raw error rate: ≤ 5%) | 70,000 | 1,350,000 | ~12 |
| Single molecule, real time sequencing by synthesis (Pacific BioSciences/Cornell Univ.) | Third | 1000–1500 | 99.3% at 15×coverage (error rate of a single read: 15–20%) | – | – | <1 |
| Nanopore sequencing (Oxford Nanopore Technologies/Harvard Univ.) | Third | Potentially unlimited? | -- | -- | -- | >20 |
| Transmission electron microscopy single-molecule sequencing (ZS Genetics, Halcyon Molecular) | Third | Potentially unlimited? | -- | ~10,000 | ~1,000,000 | ~14 |