Fast DNA Sequencing Research Articles

Using Graphdiyne Nanoribbons for Molecular Electronics Spectroscopy and Nucleobase Identification: A Theoretical Investigation.

In pursuit of fast, cost-effective, and reliable DNA sequencing techniques, a variety of two-dimensional (2D) material-based nanodevices such as solid-state nanopores and nanochannels have been explored and established. Given the promising potential of graphene for the design and fabrication of nanobiosensors, other 2D carbon allotropes such as graphyne and graphdiyne have also attracted a great deal of attention as candidate materials for the development of sequencing technology. Herein, employing the 2D electronic molecular spectroscopy (2DMES) method, we investigate the capability of graphdiyne nanoribbons (GDNRs) as the building blocks of a feasible, precise, and ultrafast sequencing device. Using first-principles calculations, we study the adsorption of four canonical nucleobases (NBs), i.e., adenine (A), cytosine (C), guanine (G), and thymine (T) on an armchair GDNR (AGDNR). Our calculations reveal that compared to graphene, graphdiyne demonstrates more distinct binding energies for different NBs, indicating its more promising ability to unambiguously recognize DNA bases. Utilizing the 2DMES technique, we calculate the differential conductance (Δg) of the studied NB-AGDNR systems and show that the resulting Δg maps, unique for each NB-AGDNR complex, can be used to recognize each individual NB without ambiguity. We also investigate the conductance sensitivity of the proposed nanobiosensor and show that it exhibits high sensitivity and selectivity toward various NBs. Thus, our proposed graphdiyne-based nanodevice would hold promise for next-generation DNA sequencing technology.

Fast DNA-analyses for surveillance of microbial communities in full-scale deammonification tanks: Potential for control and troubleshooting

The partial nitritation/anammox process is a popular process for sidestream nitrogen removal, but the process is sensitive to disturbances and requires extensive surveillance and monitoring for optimal performance. We followed two parallel sidestream full-scale deammonification reactors treating digester centrate for a year with high time-resolution of both online sensor data and microbial community as measured by Nanopore DNA sequencing. DNA surveillance revealed system disturbances and allowed for detection of process and equipment upsets, and it facilitated remediating operational actions. Surveillance of anammox bacteria (Ca. Brocadia) revealed unexpected variations, and the composition and dynamics of the flanking community indicated causes for occasional process disturbances with poor nitrogen removal. Monitoring the ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) could potentially allow reactor operation with increased dissolved oxygen (DO), yielding higher ammonia conversion while keeping NOB in control. The use of fast and frequent DNA sequencing (sampling 3–5 times a week, analysed once per week) was an important supplement, and in many cases superior, to the online sensor data for process surveillance, understanding and control.

Challenges for precision public health communication in the era of genomic medicine

Although still in the early stages of development, the advent of fast, high-output, and cost-effective next-generation DNA sequencing technology is moving precision medicine into public health. Before this shift toward next-generation sequencing in public health settings, individual patients met geneticists after showing symptoms and through limited family screening. In the new era of precision public health, everyone is a possible participant in genetic sequencing, simply by being born (newborn screening), by donating blood (biobanking), or through population screening. These initiatives are increasingly offered to individuals throughout their life and more individuals are encountering opportunities to use DNA sequencing. This article raises awareness of these growing areas and calls for different models of public engagement and communication about genomics, including screening asymptomatic populations, obtaining consent for unspecified and unforeseen future uses of genomic data, and managing variants of uncertain significance. Given that such communication challenges loom large, established norms of practice in genomic medicine and research should be reconsidered.

Interband plasmon-enhanced optical absorption of DNA nucleobases through the graphene nanopore.

We propose a novel, to the best of our knowledge, plasmonic-based methodology for the purpose of fast DNA sequencing. The interband surface plasmon resonance and field-enhancement properties of graphene nanopore in the presence of the DNA nucleobases are investigated using a hybrid quantum/classical method (HQCM), which employs time-dependent density functional theory and a quasistatic finite difference time domain approach. In the strong plasmonic-molecular coupling regime where the plasmon and DNA absorption frequencies are degenerated, the optical response of DNA molecule in the vicinity of the nanopore is enhanced. In contrast, when the plasmon and nucleobases resonances are detuned the distinct peaks and broadening of the molecular resonances represent the inherent properties of the nucleobase. Due to the different optical properties of DNA nucleobases in the ultraviolet (UV) region of light, the signal corresponding to the replacement of nucleobases in a DNA block can be determined by considering the differential absorbance. Results show the promising capability of the present mechanism for practical DNA sequencing.

Single nucleotide detection using bilayer MoS2 nanopores with high efficiency.

Single nucleotide detection is important for early detection of diseases and for DNA sequencing. Monolayer (ML) MoS2 nanopores have been used to identify and distinguish single nucleotides with good signal-to-noise ratio in the recent past. Here, we use a bilayer (BL) MoS2 nanopore (∼1.3 nm thick) to detect distinct single nucleotides with high spatial resolution and longer dwell time. In this study, the performance of similar sized (<3 nm) ML and BL MoS2 nanopores for detection of a single nucleotide has been compared. Both single nucleotide and single stranded DNA translocations through them are studied. For single nucleotide detection, we observe that BL MoS2 nanopores demonstrate twice the dwell time as compared to ML MoS2 nanopores with 95% confidence. Single nucleotide detection rate for BL MoS2 nanopores (50–60 nucleotides per s) is five-fold higher as compared to ML MoS2 nanopores (10–15 nucleotides per s) in 10 pM analyte concentration. For single stranded DNA, we observe 89% (for 60 DNA molecules detected) single nucleotide detection efficiency with BL MoS2 nanopores as compared to 85% for ML MoS2. The DNA sequencing efficiency through BL MoS2 nanopores is also found to be 8–10% better than through ML MoS2 nanopores, irrespective of DNA sequencing orientation. Thus, owing to improved analyte/nanopore charge interaction BL MoS2 nanopores can be used for single nucleotide detection with high resolution due to longer dwell time, detection rate and efficiency. This study demonstrates the improved ability of BL MoS2 nanopores in sequencing DNA with 8–10% higher efficiency, two-times temporally resolved single-nucleotide current signatures and five-times higher detection rate, compared to ML MoS2 nanopores.

DNA Translocation through Vertically Stacked 2D Layers of Graphene and Hexagonal Boron Nitride Heterostructure Nanopore.

Cost-effective, fast, and reliable DNA sequencing can be enabled by advances in nanopore-based methods, such as the use of atomically thin graphene membranes. However, strong interaction of DNA bases with graphene leads to undesirable effects such as sticking of DNA strands to the membrane surface. While surface functionalization is one way to counter this problem, here, we present another solution based on a heterostructure nanopore system, consisting of a monolayer of graphene and hexagonal boron nitride (hBN) each. Molecular dynamics studies of DNA translocation through this heterostructure nanopore revealed a surprising and crucial influence of the heterostructure layer order in controlling the base specific signal variability. Specifically, the heterostructure with graphene on top of hBN had nearly 3-10× lower signal variability than the one with hBN on top of graphene. Simulations point to the role of differential underside sticking of DNA bases as a possible reason for the observed influence of the layer order. Our studies can guide the development of experimental systems to study and exploit DNA translocation through two-dimensional heterostructure nanopores for single molecule sequencing and sensing applications.

DNA Sequencing with Single-Stranded DNA Rectification in a Nanogap Gated by N-Terminated Carbon Nanotube Electrodes

Fast, reliable and inexpensive DNA sequencing is an important pursuit in healthcare, especially in personalized medicine with possible deep societal impacts. Despite significant progress of various nanopore-based sequencing configurations, challenges remain in resolution (due to thermal fluctuations or to sensitivity to molecular orientation) and speed, which are calling for new approaches. Here we propose a sequencing protocol for DNA translocation through a nanopore with side-embedded N-terminated carbon nanotube electrodes. Employing DFT and Non-Equilibrium Green's Function formalism, we show that the rectification ratio (response to square pulses of alternating bias) bears high nucleobase specificity. The rectification arises due to bias-dependent resistance asymmetry on the deoxyribonucleotide-electrode interfaces. The asymmetry induces molecular charging and HOMO pinning to the electrochemical potential of one of the electrodes, assisted by an in-gap electric field effect caused by dipoles at the terminated electrode ends. This sequencing protocol is sensitive, selective with orders of magnitude, has high resolution, and it is robust to molecular orientation.

Unbalanced Ion Flushing Effect in MoS2 Nanopore Biosensors

In MoS2 nanopores, in addition to the widely measured ionic current signal to detect DNA nucleotides or epigenetic malformations, recent efforts have focused on the use of the transverse electronic current along the membrane for biomolecules sensing. This approach provides larger current magnitude and allows detection at the observed DNA translocation speed in the solid-state nanopore, which are crucial for accurate biomolecule sensing and faster DNA sequencing. As of today, no existing model is able to relate the variations of the transverse current to the interaction between ions, biomolecules and the MoS2 membrane. In this presentation, we report a comprehensive transport model based on the Poisson-Boltzmann transport formalism that considers the effect of biomolecules, electrolyte, and ion screening on conducting carriers along a MoS2 membrane. By accounting for membrane geometry and material properties used in recent experiment, we are able to extract the different electrical contributions to the transverse current variation during a DNA translocation event. In particular, we show that the major contribution to the electrical signal is caused by an unbalanced-ion-flushing effect that arises when the ion double-layer around the nanopore edge is swept out of the pore by the biomolecule translocation. By optimizing the MoS2 membrane geometry and nanopore size, we predict higher sensitivity to detect the translocation event in the corresponding transverse current signal. Thus,the identification of the unbalanced ion effect on the transverse electronic current foresees a critical new direction to improving the signal-to-noise ratio in solid-state nanopore sensing device.

P262 Dna sequencing with Scisco genetics next generation sequencing HLA typing version 5 kit

Aim Next-Generation HLA sequencing gives high resolution results, frequently without ambiguous combinations. However, it is still considered laborious and time-consuming. Scisco Genetics Version 5 kit combines indexing and exon-specific amplification in the same step, therefore the tech time is dramatically reduced to 4 h. Reduced processing time is a great advantage especially for high volume labs. Compared with other long-range PCR methods, the Scisco Genetics NGS version 5 method is much simpler and gives more flexibility. Twenty-four samples for 8 loci can be run using Illumina MiSeq Nano kit (24 h run time). Exon coverage was also increased compared with version 4 (Class I Exon 1 to Exon 7 and Class II Exon 1 to Exon 4), therefore the typing ambiguities are reduced greatly. Methods Forty-seven samples with known HLA types were tested using Scisco Genetics NGS version 5 kit on the Illumina MiSeq instrument. Forty-three DNA samples were isolated from blood, one sample from buccal swabs, and three samples from saliva. Two samples with rare HLA types were included in the sample population. The test data was analyzed using GeMS HLA v65.20 analysis software and the typing results were compared with the previous typing data produced by Scisco Genetics NGS version 4 kit and SBT. Results were compared for accuracy and reproducibility of HLA typing, and the ability to detect rare alleles. Presence of preferential amplification, allelic dropout, and null alleles were also evaluated. Results Of the forty-seven samples tested, forty-one samples showed results that are 100% concordance with the previous data obtained by Scisco version 4 kit. The two rare types (C∗12:143 and DRB1∗11:129) were both detected with the version 5 kit. Allele dropout was observed in two samples, and three samples had failure in one or more loci. We did not get any typing from buccal swab DNA, as was the case with previously used methods. Intronic sequence for common null alleles is also included in the analysis software. No null allele population was detected. Conclusions In summary, fast high resolution DNA sequencing results, with few ambiguities, can be produced utilizing the Scisco Genetics Version 5 NGS HLA Typing Kit.

Full-Length Mitochondrial-DNA Sequencing on the PacBio RSII.

Conventional mitochondrial-DNA (MT DNA) sequencing approaches use Sanger sequencing of 20-40 partially overlapping PCR fragments per individual, which is a time- and resource-consuming process. We have developed a high-throughput, accurate, fast, and cost-effective human MT DNA sequencing approach. In this setup we first generate long-range PCR products for two partially overlapping 7.7 and 9.2 kb MT DNA-specific amplicons, add sample-specific barcodes, and sequence these on the PacBio RSII system to obtain full-length MT DNA sequences for genotyping/haplotyping purposes.

Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography

Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples.

Development and evaluation of a novel fast broad-range 16S ribosomal DNA PCR and sequencing assay for diagnosis of bacterial infective endocarditis: multi-year experience in a large Canadian healthcare zone and a literature review.

BackgroundThe study aimed to explore the sensitivity and specificity of a novel fast 16S rDNA PCR and sequencing assay for the improved diagnosis of infective endocarditis (IE) in patients with suspected native or prosthetic heart valve (HV) infection over a multi-year period at our cardiovascular center.MethodsSixty-eight patients were prospectively enrolled who underwent HV replacement for suspected or confirmed IE between February 1, 2009 and September 1, 2014. Patient demographics, medical co-morbidities, Duke’s criteria, culture results, and antibiotic therapy were collected by detailed chart reviews. Dual-priming oligonucleotide primers targeted to 500 bps of the V1-V3 region of the 16S rRNA gene were used to perform fast broad-range 16S rDNA PCR and Sanger sequencing on ribosomal DNA extracted from HV tissues. The performance/diagnostic efficiency of the molecular test was evaluated against blood cultures and Gram stain and culture of HV tissue in patients’ with definite IE according to Duke’s criteria.ResultsFifty patients (73.5 %) had definite IE and another 8 (11.8 %) had possible IE according to Duke’s criteria. Cardiac surgery was delayed an average of 15.4 days from the time of the patient’s last positive blood culture, and appropriate antibiotic therapy was given in the pre-operative period. While 44/50 (88 %) patients had a positive blood culture, HV tissue culture was only positive in 23 (46 %) of them. Molecular testing of all HV tissues had sensitivity, specificity, NPV and PPV of 92, 77.8, 77.8 and 92 % compared to 44, 100, 39.1 and 100 % respectively for culture for diagnosis of definite IE. For prosthetic HV tissue, 16S rDNA PCR had sensitivity of 93 % and specificity of 83 % compared to 35 and 100 % respectively for culture. A literature review showed that the diagnostic accuracy of our novel fast broad-range 16S rDNA PCR assay was similar or better than that of previously published studies.ConclusionsThis novel fast broad-range 16S rDNA PCR/sequencing test had superior sensitivity compared to tissue Gram stain and culture for identifying underlying bacterial pathogen in both native and prosthetic valve endocarditis.

Side-gated ultrathin-channel nanopore FET sensors

A side-gated, ultrathin-channel nanopore FET (SGNAFET) is proposed for fast and label-free DNA sequencing. The concept of the SGNAFET comprises the detection of changes in the channel current during DNA translocation through a nanopore and identifying the four types of nucleotides as a result of these changes. To achieve this goal, both p- and n-type SGNAFETs with a channel thicknesses of 2 or 4 nm were fabricated, and the stable transistor operation of both SGNAFETs in air, water, and a KCl buffer solution were confirmed. In addition, synchronized current changes were observed between the ionic current through the nanopore and the SGNAFET’s drain current during DNA translocation through the nanopore.

Graphene nanodevices for DNA sequencing.

Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with nanopores. Owing to its unique structure and properties, graphene provides interesting opportunities for the development of a new sequencing technology. In recent years, a wide range of creative ideas for graphene sequencers have been theoretically proposed and the first experimental demonstrations have begun to appear. Here, we review the different approaches to using graphene nanodevices for DNA sequencing, which involve DNA passing through graphene nanopores, nanogaps, and nanoribbons, and the physisorption of DNA on graphene nanostructures. We discuss the advantages and problems of each of these key techniques, and provide a perspective on the use of graphene in future DNA sequencing technology.

An interview with Charles Cantor: achievements and advice from 50 years of biotechnology expertise

Interview with Professor Charles Cantor PhD by Claire Raison (Commissioning Editor)Professor Charles Cantor was awarded his PhD from the University of California, Berkeley (CA, USA), in 1966, and via professorships at Columbia University (New York, NY, USA), UC Berkeley and Boston University (Boston, MA, USA), went on to be Chief Scientific Officer of Sequenom (San Diego, CA, USA) and now retired, continues to act as consultant for more than 16 biotechnology companies. His research focused on inventing new techniques for solving biological problems, including faster DNA sequencing, better DNA amplification and advanced methods: for nucleic acid separation.

A graphene field-effect transistor as a molecule-specific probe of DNA nucleobases.

Fast and reliable DNA sequencing is a long-standing target in biomedical research. Recent advances in graphene-based electrical sensors have demonstrated their unprecedented sensitivity to adsorbed molecules, which holds great promise for label-free DNA sequencing technology. To date, the proposed sequencing approaches rely on the ability of graphene electric devices to probe molecular-specific interactions with a graphene surface. Here we experimentally demonstrate the use of graphene field-effect transistors (GFETs) as probes of the presence of a layer of individual DNA nucleobases adsorbed on the graphene surface. We show that GFETs are able to measure distinct coverage-dependent conductance signatures upon adsorption of the four different DNA nucleobases; a result that can be attributed to the formation of an interface dipole field. Comparison between experimental GFET results and synchrotron-based material analysis allowed prediction of the ultimate device sensitivity, and assessment of the feasibility of single nucleobase sensing with graphene.

Surface modification of solid-state nanopores for sticky-free translocation of single-stranded DNA.

Nanopore technology is one of the most promising approaches for fast and low-cost DNA sequencing application. Single-stranded DNA detection is primary objective in such device, while solid-state nanopores remain less explored than their biological counterparts due to bio-molecule clogging issue caused by surface interaction between DNA and nanopore wall. By surface coating a layer of polyethylene glycol (PEG), solid-state nanopore can achieve long lifetime for single-stranded DNA sticky-free translocation at pH 11.5. Associated with elimination of non-specific binding of molecule, PEG coated nanopore presents new surface characteristic as less hydrophility, lower 1/f noise, and passivated surface charge responsiveness on pH. Meanwhile, conductance blockage of single-stranded DNA is found to be deeper than double-stranded DNA, which can be well described by a string of blobs model for a quasi-equilibrium state polymer in constraint space.

The promise of nanopore technology: Nanopore DNA sequencing represents a fundamental change in the way that genomic information is read, with potentially big savings.

For those following DNA sequencing trends closely, nanopores have been something of a buzzword for a number of years, representing a theoretical platform for fast, cheap, and ubiquitous DNA sequencing. Nanopore sequencing is now becoming a reality, but since the concept was introduced, other technologies have reduced sequencing costs by several orders of magnitude, raising the bar for success.

Interview with Donald E Ingber.

Interview with Donald E Ingber.

Lysosomal diseases: diagnostic update

Technological developments in newborn and population screening, biomarker discovery for monitoring treatment and rapid high throughput DNA sequencing are having a great impact on the diagnostic procedure for symptomatic patients with lysosomal storage diseases. The use of dried blood spots, initially for newborn screening, has stimulated the introduction of automated, rapid and more sensitive methods for the assay of lysosomal enzymes, including the synthesis of novel substrates. Storage products and secondary metabolites in urine and cells can be identified and measured very accurately and sensitively by high performance liquid chromatography and tandem mass spectrometry. This has enhanced the preliminary metabolite screen for LSDs and facilitated the diagnosis of transport defects. Fast, reliable and affordable high throughput DNA sequencing, such as whole or selected exome sequencing, is helping to make diagnoses in difficult cases, to reveal novel gene defects, to widen the clinical spectrum of diseases and possibly to identify modifying genetic factors. Bioinformatics will be necessary to handle the data generated by these new technologies. Notwithstanding, these technical innovations, accurate and reliable diagnosis will still depend on the knowledge and experience of skilled laboratory staff.