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Advances in sequencing have transformed the way scientist think about and solve problems on a genetic level. Genomic analysis has yielded insight into the causes and cures of disease and environmental factors affecting health, complex ecological interactions, and evolution, among other scientific questions. Sequencers are used to diagnose disease and screen for the presence of genetic risk factors, though, clinical applications are certainly not the only application for today’s sequencing technologies. As advances in sequencing technology have expanded, so have the applications.
Until now, however, no such advancement has redefined how sequencing is thought of as a tool.
Third-generation technologies have started to change this fact. At the head of the third-generation technology race is Pacific Bio Sciences’ (PacBio) SMRT sequencers and Oxford Nanopore Technologies’ MinIon nanopore sequencer. Though PacBio boasts slightly higher consensus accuracy, the MinIon presents greater potential for novel applications. Sequencing capability combined with the portability of the MinIon has allowed scientists to use sequencing in new and more creative ways.
Initially a slow process, our understanding of the significance of DNA wasn’t realized until the Hershey-Chase experiments in 1952 which led to the discovery that it is DNA that carries genetic information for a cell, rather than protein (Timeline 2016). With this discovery, the rate of advances in genetics increased exponentially and it wasn’t long before it became clear that knowing the sequence of nucleotides was a necessity to fully understand the complexities of genetics. Thus, the development of genetic sequencing began, marking a new revolution in biology.
The revolution began with the development of primitive RNA sequencing methods, first used by Walter Fiers, to sequence the original genome of the Bacteriophage MS2 (Schiller 2010). One of the first DNA sequences was just 24 base pairs long and used a method known as wandering-spot analysis (Schiller 2010). This cumbersome and slow method highlights just how far modern methods have come. Following this method came Sanger and Coulson’s “plus-minus” method and shortly after this came the Maxam-Gilbert sequencing method though neither were ideal for ease-of-use biology kits and thus could not be scaled to the entire genetics community. These issues were soon surpassed with Frederick Sanger’s development of the chain-termination sequencing method.
Sanger sequencing utilizes dideoxynucleotide triphosphates in solution with deoxynucleotide triphosphates to terminate a DNA sequence at a random nucleotide. Each type of dideoxynucleotide is labeled with a specific fluorescent tag, allowing the base added to be discerned. This is done across thousands of random fragments and is resolved using capillary electrophoresis, a gel electrophoresis method yielding a resolution of one base pair (Hagemann 2015). Coupled with a hierarchical shot gun sequencing method, sanger sequencing provided the initial sequences in the human genome project, though methods varied slightly between participating centers (International 2001). Although pivotal in the advance of sequencing, Sanger technology is limited due to relatively short read length and low throughput. These limitations are best depicted by the near 8-fold increase in sequence output in the human genome project, following the instillation of more advanced sequence detectors (International 2001).
However, sanger sequencing has continued to be useful for specific applications as it is simple and highly cost effective for short-read uses. One still current use for sanger is in clinical applications where a high volume of samples can be run at low cost to detect gene-specific mutations. Dr. Albitar and his team at NeoGenomics Laboratories in Irvine, CA describe the use of Sanger sequencing in a high sensitivity assay for the detection of resistance mutations responsible for a loss of therapeutic activity of bruton tyrosine kinase inhibitors (Albitar 2017). Using their high-sensitivity sanger sequencing, Albitar and colleagues were able to lower the limit of detection for sanger sequencing to 1 mutant out of 1000 WT alleles (0.1%) and thus detect low frequency mutant populations (Albitar 2017). However, the same high sensitivity sequencing assay was also applied to next generation sequencing technology (Albitar 2017). This suggests that the researchers acknowledge the growing utility of next generation sequencing in clinical applications as costs of next-gen technology continue to fall and machines require less prep-work.
The next advance in sequencing technology to make a significant and lasting effect came with the development of paired-end sequencing by synthesis and the Illumina sequencing platforms. The Illumina genome analyzer marked a vast increase in sequence throughput, thus becoming the tech of choice for whole-genome sequencing (Shokralla 2012). Illumina sequencing uses sequencing by synthesis on bridge-amplified DNA fragments attached to a flow cell. The flow cell is covered with adapters complimentary to specific adapter sequences ligated to the DNA fragments to be sequenced. When the DNA fragments are added, the complimentary sequences bind, and the lawn of fragments is expanded through bridge amplification resulting in clusters of millions of fragments. The sequencing steps begin with the addition of the four nucleotides, each labeled with a fluorescent probe at the 3’ OH (thus blocking elongation). After addition of the correct nucleotide the probe is excited and imaged. Next, a wash cycle chemically cleaves the fluorescent probe so another fluorescently labeled nucleotide can be added (Shokralla 2012). This is done in parallel across millions of fragments, producing millions of short reads of an entire genome that can then be pieced together using one of the multiple Illumina sequence software options (Sequencing Software 2018).
The primary advantage of Illumina sequencing is the extremely high throughput resulting in faster sequencing and lower cost per base pair sequenced. Additionally, due to the fragment amplification/cluster generation step, Illumina sequencing provides greater resolution when searching for representative sequences in a pool compared to previous methods; an especially helpful tool in the identification of viruses or microbes in an environmental sample.
Iaconelli and colleagues at the Department of Environment and Health in Rome, Italy demonstrated this fact by using a MiSeq II sequencer (Illumina) to characterize human adeno viruses found in raw sewage samples. Initially, the researchers used nested PCR (to specifically amplify the hypervariable regions of the hexon gene found in all human adeno viruses) coupled with Sanger sequencing (Iaconelli 2017). This resulted in the identification of 4 human adenovirus types, corresponding to 4 different species, and 26 amplicons that were uncharacterizable (Iaconelli 2017). Following resequencing of these types using Illumina, an additional 15 types and 2 species of human adenovirus were characterized (Iaconelli 2017). These findings expand the application potential of next generation sequencing technologies like Illumina to the study of pathogens in environmental water. However, for this application and many others, the technology still has considerable limitations.
The dominant short-coming with Illumina technology is the short fragment size resulting in short reads. This is the result of dephasing and optical signal decay as well as the appreciable increase in error rate as sequence reads get longer (Shokralla 2012). These short reads are unable to span regions of heterozygosity, segmental duplications, GC bias and diverse families of repeats in the genome, making the accurate assembly of these reads extremely difficult and, in some regions, impossible, even if a reference genome can be used (Jain 2018). Though upgrades in the Illumina platform like Hiseq have been developed, this limitation still exists as read lengths are at a maximum of 150 base pairs. In addition, the need for sample preparation and fragment amplification limit utility when compared to more advanced technologies like nanopore sequencing.
The MinIon sequencer from Oxford Nanopore Technologies is a pocket-sized sequencer weighing about 90 grams. The device consists of a flow cell attached to 2048 nanopores that can be addressed individually (Jain 2016). Before sequencing, the DNA or cDNA fragments have adapters ligated to the ends. The 5’ adapter facilitates loading of the strand into a processive enzyme attached to the nanopore. The 3’ adapter depends on the type of sequencing being conducted. The 2D read function of the MinIon uses a hair-pin adapter at the 3’ end to link the complementary strand to the leading strand thus allowing the nanopore to read both strands in one process (Jain 2016). This results in higher accuracy but sacrifices higher through put. 1D sequencing on the MinIon does not use a 3’ hair-pin adapter. This results in longer read lengths, higher throughput and shorter prep time, though it sacrifices accuracy.
Sequencing occurs as the processive, helicase-type enzyme, attached at the opening of the nanopore, unzips the dsDNA fragment and ratchets it through the pore. The nanopore has an electric potential run through its narrowest point. As sets of nucleotides pass through the pore, they cause a characteristic disruption in the electric potential which is detected by a sensor. These characteristic disruptions are then interpreted as a sequence of 3-6 nucleotides (Jain 2016).
The chief advancement provided by third-generation sequencers like the MinIon is the capability of longer reads and real-time results. The longer reads can span complex regions of the DNA that scientists were not previously able to sequence due to the reasons mentioned above. Therefore, long reads are advantageous when no reference genome is available or when constructing a reference genome.
Dr. Miten Jain and colleagues at the university of California, Santa Cruz Genomics Institute used the ultra-long read capability of the MinIon nanopore sequencer (Oxford Nanopore Technologies) to sequence and assemble a reference genome for the human GM12878 Utah/Ceph cell line. The researchers sequenced DNA directly, with out PCR amplification, and reported an N50 of 10,589 bp and a median coverage depth of 26-fold for initial sequencing (Jain 2018). The researchers then produced ultra-long reads (N50 > 100 kb) by saturating the Oxford Nanopore Rapid kit with high molecular weight DNA, getting reads with an N50 of 99.7 kb (Jain 2018). With the de novo-assembled sequence, the researchers were able to close 12 gaps in the human genome assembly that were greater than 50kb in size (Jain 2018). The assembly closed gaps of tandem repeats and segmental duplications and 17 ultra-long reads containing telomeric tandem repeats were successfully mapped back to specific chromosome assemblies (Jain 2018).
The researchers demonstrated that nanopore technology can be used in the de novo assembly of a genome. It was also demonstrated that long and ultra-long reads produced by the MinIon sequencer are useful in filling in gaps that previous, shorter-read technologies such as Illumina are incapable of doing. However, sequencing accuracy still falls short of shorter read technologies and the base-calling algorithm for whole genome assembly using the MinIon requires further development as call bias is still present (Jain 2018). Thus, for this type of application, nanopore sequencing is not yet ready to completely replace technologies like Illumina. Rather, it is a powerful tool that can be used in complementarity with short read technologies and thus does not significantly re-define sequencing as a tool.
One way in which Oxford nanopore has changed the way sequencing as a tool is used is through its portability, ability to produce real-time results, and its direct sequencing capability. The real-time utility of the MinIon has allowed for functions like “Read Until.” This function, developed by Loose and colleagues, can screen for a specific target sequence (Jain 2016). As each fragment is read from the flow cell, the resultant sequence is aligned to the target sequence (Jain 2016). Each fragment continues to be read by the nanopore as long as the resultant sequence matches the target sequence. If the fragment does not match the target sequence, it is ejected from the nanopore and the process begins again. This results in a rapid accumulation of target strand reads (Jain 2016). The accumulation of reads, increasing targeted read depth, also serves to compensate for lower accuracy. In one study, researchers found the theoretical probability for one or more miscalled base in a genome sequenced using the MinIon was less than 5% when the read depth in every position was greater than 33 (Hoenen 2016).
The “Read Until” function significantly reduces the total time required to sequence a sample making the MinIon ideal for clinical applications. Furthermore, its small size and streamlined prep time expands MinIon utility to remote applications. Thomas Hoenen and collaborators successfully used the MinIon to obtain Ebola genome sequences from infected patients at a field diagnostic lab in Liberia. The researchers reached a capacity of four full-length genomes per day using two MinIon sequencers and a single laboratory worker (Hoenen 2016). Genome sequences obtained during an outbreak are key for the identification of virus evolution patterns and exploring chains of transmission (Hoenen 2016).
In addition to remote analysis of clinical samples, the in-field analysis of environmental samples is another new application where the MinIon has demonstrated utility. Arwyn Edwards from the Institute of Biological, Environmental and Rural Sciences at Aberystwyth University and his team of collaborators used the MinIon sequencer to characterize supraglacial microbiota on the Greenland Ice Sheet and in the Austrian Alps. Edwards and his team extracted genomic DNA directly from a darkly colored, microbe-mineral aggregate found on glaciers called cryoconite and characterized the microbiome using both shotgun metagenomics and 16S rRNA approaches (Edwards 2018). This protocol was conducted at a minimalistic field laboratory using a range of preparation methods (Edwards 2018). The method conducted in the most extreme setting was conducted without a thermocycler and no power (Edwards 2018). Instead the researchers used body-warmed reagents, an insulated MinIon sequencer and “[held the] library tube in gloved hands for the 30°C incubation step and [immersed it] in hot water stored in an insulated mug for 75°C inactivation” for library preparation (Edwards 2018).
Even with extreme environmental factors, 796 reads of 2372 reads sequenced were successfully matched to taxonomy (Edwards 2018). Samples prepared under more favorable conditions (i.e. Power, mini thermocyclers, and cloud-based bioinformatic tools) assigned 2305 of 3514 reads to named taxa for the metagenomics experiments (Edwards 2018). 16S results, on the other hand, suffered due to high error rate and limited number of barcodes that could be amplified (Edwards 2018). Even so, the study demonstrates the potential of nanopore technology as a viable tool for remote, in-field characterization of microbiota in environmental samples.
Vast advances have been made in sequencing technology, leading to even greater discoveries in the field of genomics. As sequencing technology has improved, researchers have been able to use it in an increasing number of applications. At the start of the sequencing era, accuracy and throughput were the primary issues of concern for the technology. Now, with these issues largely satisfied, the most ground-breaking advances in sequencing come in the form of long reads and ease of use. This is where Oxford nanopore excels.
Previously, sequencing was thought as a laboratory tool to be used only after weeks of preparation and consideration because that’s what cost, both of money and time, required. Currently, there are many sequencing technologies that have lowered the cost barrier thus allowing more free and innovative use of sequencing technology. Although, only the MinIon has been able to take the technology out of the laboratory. Its small size, yielding extreme portability, no required calibration or setup, and fast data turn around, make the MinIon well suited for in-field analysis of clinical and environmental samples therefore bypassing the need for isolation and cultivation of specimens in a laboratory. This saves both time and money, resulting in faster, more efficient science and changing the way sequencing can be used as a tool.
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