This is the world’s only human genome microarray based on the completely sequenced human genome. H25K is a multipurpose long oligonucleotide microarray that allows karyotyping, gene expression profiling, chromatin structure analysis, and protein–DNA interaction studies on a genomic scale. Its glass substrate slide format is fully compatible with every major microarray scanner brand including the Arrayit InnoScan and SpotLight Scanner series.

Although genome-wide association studies have successfully identified associations of many common SNPs with common diseases, the SNPs implicated so far account for only a small proportion of the genetic variability of tested diseases. It has been suggested that common diseases may often be caused by rare alleles missed by genome-wide association studies. High-throughput, high-accuracy resequencing technologies needed to identify these rare alleles. Although array-based genotyping has allowed genome-wide association studies of common SNPs in tens of thousands of samples, array-based resequencing has been limited for two main reasons: the lack of a fully multiplexed pipeline for high-throughput sample processing and failure to achieve sufficient performance. Scientists at Affymetrix and Genentech in collaboration with Stanford Genome Technology Center have solved both of these problems and created a fully multiplexed high-throughput pipeline that results in high-quality data (Zheng et al. 2009). The pipeline consists of target amplification from genomic DNA, followed by allele enrichment to generate pools of purified variant (or nonvariant) DNA, and ends with interrogation of purified DNA on resequencing arrays. They have used this pipeline to resequence ≈5 Mb of DNA (on three arrays) corresponding to the exons of 1,500 genes in >473 samples; in total >2,350 Mb were sequenced. In the context of this large-scale study, they obtained a false-positive rate of ≈1 in 500,000 bp and a false-negative rate of ≈10 %. Some of the advantages of this approach are:

Strand-specific RNA sequencing is rapidly replacing conventional cDNA sequencing as an approach for assessing information about the transcriptome. Alongside improved laboratory protocols, the development of bioinformatic tools is steadily progressing. Currently the Illumina TruSeq library preparation kit is used, along with additional reagents, to make stranded libraries in an automated fashion, which are then sequenced on Illumina HiSeq 2000. By use of freely available bioinformatic tools, it was shown through quality metrics that the protocol is robust and reproducible (Sigurgeirsson et al. 2014). This study further highlights the practicality of strand-specific libraries by comparing them to non-stranded libraries, by looking at known antisense transcription of pseudogenes, and by identifying novel transcription. Non-stranded, Illumina TruSeq kit can be adapted to generate strand-specific libraries and can be used to access detailed information on the transcriptome. The Ribo-Zero kit is very effective in removing ribosomal RNA from total RNA, and the STAR aligner produces high mapping yield in a short time. Strand-specific data gives more detailed and correct results than do non-stranded data as was shown when estimating expression values and in assembling transcripts. Even well-annotated genomes need improvements and corrections, which can be achieved using strand-specific data. It is recommended that researchers in this area should use strand-specific data as it provides more confidence in the data analysis and is less likely to lead to false conclusions. If faced with analyzing non-stranded data, researchers should be well aware of the caveats of that approach.

Human exome sequencing is considered to be “the holy grail” of resequencing studies that will ultimately lead to significant biomedical breakthroughs. Exomes are the parts of genes that encode amino acids. As such, exome sequencing could enable the discovery of much of the CNV that is responsible for many common and rare diseases (e.g., cancer and Alzheimer’s disease). Exome resequencing is also expected to possibly shed light on why some diseases occur more often in certain populations and could help uncover why drugs are effective only in a subset of the individuals or population. Applications of array-based whole-exome sequencing (WES) include some rare genetic disorders. Exome sequencing will ultimately produce technology to feed the research pipeline and nourish the development of personalized healthcare. However, the sequencing of the exome by conventional PCR methods is neither technically nor economically feasible because the preparation of human coding exons is very expensive and time consuming.

Human exome microarrays have made human exome sequencing feasible. These high-density arrays of long oligonucleotide probes provide greater information content and higher data quality necessary for studying the full diversity of genomic and epigenomic variation. The enhanced performance is made possible by Maskless Array Synthesis (Roche) technology, which uses digital light processing and rapid, high-yield photochemistry to synthesize long oligonucleotide, high-density DNA microarrays with extreme flexibility.

Human Exon 1.0 ST Array (Affymetrix) is used for expression profiling at both gene and exon levels. Although it provides accurate assessments of gene expression, only 20 % of its probe sets are supported by high-confidence annotations, and each probe set contains only one to four probes. As a result, the array is susceptible to false-positives for analysis of exons and alternative splicing. An alternative design is to use probes targeting exon–exon junctions in addition to exons, which has been applied to several custom arrays. However, further improvements are needed for cost-effective high-throughput applications in clinical trials.

An optical technique has been developed for the parallel manipulation of nanoscale structures with molecular resolution (Csaki et al. 2007). Bioconjugated metal nanoparticles are positioned at the location of interest, such as certain DNA sequences along metaphase chromosomes, prior to pulsed laser light irradiation of the whole sample. Nanoparticles are designed to absorb the introduced energy highly efficiently, thus acting as nanoantenna. As result of the interaction, structural changes of the sample with subwavelength dimensions and nanoscale precision are observed at the location of the particles. The process leading to the nanolocalized destruction is caused by particle ablation as well as thermal damage of the surrounding material. The procedure is highly parallel and can be potentially multiplexed by addressing several different sequences (such as genes). Potential applications are in DNA analysis such as DNA fingerprinting or mutation analysis as well as single-molecule manipulation.

By partially denaturing YOYO^®-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, a sequence-dependent “barcode” was obtained corresponding to a series of local dips and peaks in the intensity trace along the extended molecule (Reisner et al. 2010). This structure arises from the physics of local denaturation and statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.

The translocation of polymers across nanometer-scale apertures in cell membranes is a common phenomenon in biological systems. The change in electrical conductance of a single nanopore as a polymer transits the pore can be reliably detected and used to characterize the polymer. As saline solution flows through the nanopore, it creates an electric current. However, when a DNA molecule passes through the pore, the current is disrupted and the amount of current disruption depends on which of the nucleobases adenine (A), thymine (T), cytosine (C), or guanine (G) is in the pore. To read the sequence of nucleobases, a scientist simply has to find out how much each base disrupts the electric current. This information enables one to read the sequence of DNA bases simply by logging the sequence of electrical disruptions as a DNA molecule passed through.

It is possible to refine this method to the point where the base sequence of a DNA strand can be read with single-base resolution as the DNA transits the pore, which measures 2–5 nm. This would provide a sequencing method that is faster and cheaper than existing ones by many orders of magnitude (Ghosal 2007). A method of identifying single molecules that does not rely on expensive and complex fluorescent labeling is important for reducing the cost and increasing the speed of genome analysis. It is possible to achieve this goal by monitoring a simple electric current passing through a nanopore. As single DNA bases pass through the nanopore, each base causes a characteristic disruption of current that allows the molecule to be identified. A study has shown that this system can also directly identify methylated cytosine, which can be distinguished from the four DNA bases using the same method (Clarke et al. 2009).

Controlling DNA translocation speed is critically important for nanopore sequencing as free electrophoretic threading is far too rapid to resolve individual bases. A number of promising strategies have been explored in recent years, largely driven by the demands of NGS. Engineering DNA–nanopore interactions with organic coatings is an attractive method as it does not require sample modification, processive enzymes, or complicated and expensive fabrication steps. For the first time, fourfold tuning of unfolded, single-file translocation time through small, amine-functionalized solid-state nanopores has been demonstrated by varying the solution pH in situ (Anderson et al. 2013). Additionally, the authors developed a simple analytical model based on electrostatic interactions to explain this effect which will be a useful tool in designing future devices and experiments. This method of reading DNA sequences is still in development. A nanopore chip could be built into a portable device that could be brought right to the patient at the POC.

Hybridization-Assisted Nanopore Sequencing (NABsys) platform combines nanopore sequencing with sequencing by hybridization. Unlike other nanopore-based sequencing approaches, the NABsys platform does not depend on single-base resolution of the nanopore detector in order to obtain accurate sequence information.

Preliminary findings have been described on the use of Mycobacterium smegmatis porin A (MspA) for nanopore sequencing (Derrington et al. 2010). Based on the short, narrow nature of the MspA pore, it is considered better than commonly used alpha-hemolysin protein for nanopore-sequencing applications. It is just short enough to accommodate one or two nucleotides. MspA-based protein channels could be used to distinguish between all four nucleotides in ssDNA based on ion current signals and resolve single nucleotides in ssDNA when dsDNA temporarily holds the nucleotides in the pore constriction. By tossing in DNA hairpins or dsDNA, it is possible to slow DNA progression through the pores so that this nucleotide sequence can be observed. It is also possible to genetically engineer MspA to optimize the constriction zone for nanopore sequencing. For example, a mutant version of the MspA was designed that has a neutral, uncharged constriction zone allowing charged ssDNA to move through it unhindered. Similarly, subsequent experiments suggest that punctuating ssDNA with double-stranded sequence, duplex-interrupted nanopore sequencing, can also slow nucleotide movement through the channel to aid sequencing, since dsDNA tends to get stuck in the pore and cannot pass through until dissociated. The ultimate aim is to develop an MspA-based nanopore-sequencing strategy that can unravel sequence of DNA that has not been modified and is cheap as well as easy to perform. For this purpose other strategies for slowing ssDNA movement through the channels are being devised and tested. This approach has been patented and is expected to become commercially available.

IBM Research’s DNA Transistor technology offers true single-molecule sequencing by decoding molecules of DNA while they are threaded through a nanometer-sized pore in a silicon chip. Ultimately, the technology could improve throughput and reduce costs to where the whole human genome can be sequenced at a cost of between $100 and $1,000.

In nanopore strand sequencing, a single strand of DNA moves through a narrow pore and the bases are identified as they pass a reading head (Cockroft et al. 2008). This is a rapid real-time technology, which is the cornerstone of a number of advanced single-molecule DNA-sequencing concepts and does not require the time-consuming cyclic addition of reagents. After implementing a chip with a million pores, the researchers expect nanopore sequencing to achieve a 15-min genome by end of 2014 with a very short sample preparation time.

Although the DNA sequence information obtained from nanopores comes from the signal collected during DNA translocation, the throughput of the method is determined by the rate at which molecules arrive and thread into the pores. The process of DNA capture into nanofabricated SiN pores of molecular dimensions has been investigated (Wanunu et al. 2010). For fixed analyte concentrations, there is an increase in capture rate as the DNA length increases from 800 to 8,000 base pairs, a length-independent capture rate for longer molecules, and increasing capture rates when ionic gradients are established across the pore. Furthermore, the study showed that application of a 20-fold salt gradient allows the detection of picomolar DNA concentrations at high throughput. The salt gradients enhance the electric field, focusing more molecules into the pore, thereby advancing the possibility of analyzing unamplified DNA samples using nanopores. This technology has been licensed to NobleGen Biosciences Inc. for further development.

Many of the technical hurdles preventing the use of nanopores in DNA sequencing (slowing transit through the pore, aligning bases properly for reading, designing the proper-sized nanopore) have been largely addressed. Nanoscale cover plates have been used to convert nanopores in solid-state membranes into versatile devices for label-free molecular sensing, and the custom apertures in the nanoplates can be chemically addressed for sequence-specific detection of DNA (Wei et al. 2012). This is called “DNA origami,” i.e., the art of programming strands of DNA to fold into custom-designed structures with specified chemical properties. Different chemical components beyond DNA could be attached to the appropriate site on a DNA nanoplate.

Commercial potential of a nanopore-based sequencer is enormous—rapid speed, single-molecule analysis, no need for imaging, and tremendous scalability. Oxford Nanopore Technologies^® is developing the GridION™ system and miniaturized MinION™ device. These are a new generation of electronic molecular analysis system for use in scientific research and personalized medicine. The platform technology uses nanopores to analyze single molecules including DNA/RNA and proteins.

One problem with nanopore-based sequencing has been that DNA strands move too quickly through nanopores for electric signatures to be captured. But a technology has demonstrated the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore, Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore (Manrao et al. 2012). As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, well-resolved and reproducible ionic current levels were observed with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42–53 nucleotides long, the authors recorded current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.

INanoBio is developing a nanosensor device for high-speed genome sequencing in collaboration with other institutions. Rather than slowing down the DNA as it translocates through a nanopore, its strategy is to sequence the DNA at high speed using its FET nanosensor devices. Field-effect transistor sensors can have high switching frequencies of up to 1 GHz or more, which enables faster signal acquisition with minimal background noise. It is expected to sense of individual bases at higher speeds, with larger signal to noise ratios, than when using other strategies.

A purely electrical method has been described for the single-molecule detection of specific DNA sequences, achieved by hybridizing dsDNA with peptide nucleic acid (PNA) probes and electrophoretically threading the DNA through sub-5 nm silicon nitride pores (Singer et al. 2010). The single-molecule detection device is a solitary nanopore fabricated in a freestanding silicon nitride membrane using a focused electron beam. When a positive current is applied across the membrane using electrodes, negatively charged PNA-tagged DNA molecules are captured and guided through the nanopore. This provides control over the speed of the strand and enables researchers to identify bases by evaluating changes in ion current. The current is reduced to a value that reflects the displacement of electrolytes from the nanopore by the DNA segment. Sequence detection is performed by reading the ion current traces of individual translocating DNA molecules, which display a characteristic secondary blockade level, absent in untagged molecules. The potential for barcoding DNA has been demonstrated through nanopore analysis of once-tagged and twice-tagged DNA at different locations on the same genomic fragment.

This purely electrical method vastly improves nanopore sequencing, which is highly regarded for its potential to sequence whole genomes quickly and cheaply. Although the method solves the problem of how to detect single-nucleotide bases, which has plagued nanopore technology, it is unlikely to be used for WGS. PNAs have previously been used as part of a microfluidic approach to DNA sequencing. Sequence detection through PNA invasion of the DNA strand was problematic because it was primarily achieved through electrophoresis gel assays. Utilization of the inherent single-molecule nature of the nanopore system enables one to look at individual molecules, one by one, and judge whether or not they contain the PNA tag and, with it, the DNA sequence of interest.

This method has advantages over other single-molecule sensing methods that require the molecules to be located, which is a difficult task. With the nanopore method, long-range electric fields can be used to focus the DNA molecules from far away into the nanopore. The same electrodes that are used to generate this focusing field also induce ion flow across the pore, giving way to the ion current which is the main detection method. One can draw the DNA toward and through the nanopore by manipulating the electric field, without having to modify either the DNA strand or the pore itself. Eventually, PNA induces DNA structure change. Therefore, by detecting the PNA tags along the DNA molecule, one is in fact detecting the presence of its predefined corresponding sequence; therefore, this method is a perfect match for the nanopore system. The PNA signal is direct (enables quantification), fast, and does not require large sample sizes. This high-throughput long-read length method can be used to identify key sequences embedded in individual DNA molecules. This opens up a wide range of possibilities in human genomics as well as in pathogen detection for treating infectious diseases. Further advances in this approach may detect short DNA sequences embedded in a long DNA molecule. Thus, the single-molecule detection mechanism based on PNA-tagged ssDNA could help facilitate the use of nanopores for molecular diagnostics. Further research should explore methods geared toward detection of specific sequences for molecular diagnostics. By developing a method that searches for key sequences, one can forego the need to sequence blood samples for every disease and instead look for key molecular signatures. Given the single-molecule nature of this method, it is quite feasible that it will be possible to forego the current practice of target amplification. This would not only reduce errors but also simplify the sample preparation process, ultimately reducing costs dramatically.

Nanopatch™ technology (Electronic Biosciences) measures the activity of ion channels at the single-molecule level and can be adapted for DNA sequencing. Electronic Biosciences and its collaborators at the University of Utah have also successfully demonstrated translocation of single-stranded DNA oligomers through a protein ion channel (alpha-hemolysin) reconstituted in a lipid bilayer suspended across the quartz nanopore membranes orifice (Schibel et al. 2010). Nanopore detection of individual DNA abasic sites in single molecules was also achieved by this method (An et al. 2012).

GE Global Research (http://​ge.​geglobalresearch​.​com/​) is developing a third-generation sequencing technology that aims to be less expensive and more accurate. It is working on a means to interrogate DNA that can be used for high-throughput, single-molecule DNA sequencing. This method can utilize less expensive optics by sequencing clonally amplified DNA or, alternatively, can be developed for a high-end system that can interrogate single molecules. The proposed method is a sequencing-by-synthesis strategy. It uses two separate innovations together to accomplish DNA sequencing. First, terminal phosphate-labeled nucleotides are used with a dye attached that is incorporated and is then removed from the growing DNA strand. Next, a method was developed to “freeze” the DNA polymerase as it is incorporated into this unique nucleotide. In this state, the three-part complex of DNA strand, polymerase, and incoming base is quite stable and can be washed and interrogated to determine the identity of the trapped base by the color of the dye in the complex. After interrogation, the polymerase is allowed to add that single base and proceed on to form the complex on the next template base. This cycle is repeated over and over. The procedure takes place on a solid support in a microfluidic system. The advantages of this method are:

GE Global Research has demonstrated proof of concept for the method. It is now optimizing various aspects of its new sequencing system.

Carbon nanotubes having unique arrangements of carbon atoms exhibit many special physical and chemical properties, and researchers used these properties for DNA sequencing. ssDNA can translocate through single-walled carbon nanotube (SWCNT) with diameter of 1–2 nm (Liu et al. 2010). They fabricated devices in which one SWCNT spans a barrier between two fluid reservoirs, enabling direct electrical measurement of ion transport through the tube. A fraction of the tubes pass anomalously high ionic currents. Electrophoretic transport of small ssDNA oligomers through these tubes was marked by large transient increases in ion current and confirmed by PCR analysis. SWCNTs simplify the construction of nanopores, permit new types of electrical measurements, and may open avenues for control of DNA translocation. It is possible to slow the rate of translocation to a speed where reading the sequence may actually be possible. Researchers are refining the technique to make DNA analysis much faster and more accurate than the currently available methods.

A reader has been developed with this technology that can discriminate between DNA’s four chemical components (Chang et al. 2010). The authors constructed two electrodes, one on the end of a microscope probe and another on the surface. The end of each was chemically modified to attract and catch the DNA between a gap like a pair of chemical tweezers. The gap between these functionalized electrodes had to be adjusted to find the chemical bonding sweet spot so that when a single chemical base of DNA passed through a 2.5-nm gap between two gold electrodes, it momentarily sticks to the electrodes and a small increase in the current is detected. Any smaller and the molecules would be able to bind in many configurations, confusing the readout. Any bigger and smaller bases would not be detected. At this scale, which is just a few atomic diameters wide, quantum phenomena are at play where the electrons can actually leak from one electrode to the other, tunneling through the DNA bases in the process. Each of the chemical bases of the DNA genetic code gives a unique electrical signature as they pass between the gaps in the electrodes. It was discovered that just a single chemical modification to both electrodes could distinguish between all four DNA bases. The group is trying to adapt the reader to work in water-based solutions, which is a critically practical step for DNA-sequencing applications. Also, it will be desirable to combine reader capabilities with the carbon nanotube technology for reading short stretches of DNA.

Qdot^® nanocrystals (Life Technologies), already known for detection of individual protein molecules, has been used in SMS core sequencing engine. Compared to conventional fluorescence detection with organic dye molecules, the Qdot approach generates signals more than 100 times greater, enabling simple single-molecule detection. The SMS system uses specially designed sequencing versions of these nanocrystals, attached to proprietary DNA polymerase molecules. The system monitors the real-time incorporation of nucleotides into individual growing DNA strands. As nucleotides are incorporated, they are energized by photons transferred from the Qdot nanocrystal, generating a characteristic colored flash of fluorescent light. The prototype SMS system records the time and color series of these light flashes to determine the DNA sequence of each individual DNA strand. A key feature of this approach is that it looks for correlated fluorescence flashes as Qdot signals decrease, which should improve the error-prone profile of noisy single-molecule data.

A unique aspect of the SMS system is reagent exchange, where individual Qdot polymerases and synthesized templates can be removed and replaced with new ones. This enables immobilized individual DNA templates to be resequenced several times, enabling highly accurate reads with minimal sample preparation. In addition, reagent exchange enables linking of multiple long reads to enable virtually unlimited read lengths.

Early stage results from this SMS technology show promise to combine unlimited continuous long read lengths with unmatched accuracy for delivering targeted genomic sequence data in a matter of hours, which will facilitate adoption of sequencing as a routine tool in research laboratories as well as clinical settings. Potential applications include haplotype phasing of the human genome.

sTOP (sequencing is carried out on Top of a Photodiode) technology is developed by Crack of Taiwan with a pending patent. Upon incorporation of a nucleotide into the nascent DNA strand by the DNA polymerase at the bottom of the reaction site, the fluorophore is activated and emits light, the nature of which depends on the nucleotide. This photon signal passes through the filter layer (whereas excitation light does not), to reach the photodiode where photons are converted into an electric signal. The properties of this signal are in turn decoded by the logical circuit adjacent to the diode, leading to the identification of the incorporated nucleotide. If the DNA synthesis reaction does proceed, signals are measured, which correspond to the successive steps of nucleotide incorporation, and enable sequence determination.