DNA Sequencing Applications Research Articles

Application of cell-free DNA sequencing in characterization of bloodborne microbes and the study of microbe-disease interactions.

It is an important issue whether microorganisms can live harmoniously with normal cells in the cardiovascular system. The answer to the question will have enormous impact on medical microbiology. To address the issue, it is essential to identify and characterize the bloodborne microbes in an efficient and comprehensive manner. Due to microbial sequence complexity and the composition of significant number of unknown microbial species in the circulatory system, traditional approaches using cell culture, PCR, or microarray are not suitable for the purpose. Recent reports indicate that cell-free DNA (cfDNA) sequencing using next-generation sequencing (NGS) or single-molecule sequencing (SMS), together with bioinformatics approaches, possesses a strong potential enabling us to distinguish microbial species at the nucleotide level. Multiple studies using microbial cfDNA sequencing to identify microbes for septic patients have shown strong agreement with cell culture. Similar approaches have also been applied to reveal previously unidentified microorganisms or to demonstrate the feasibility of comprehensive assessment of bloodborne microorganisms for healthy and/or diseased individuals. SMS using either SMRT (single-molecule real-time) sequencing or Nanopore sequencing are providing new momentum to reinforce this line of investigation. Taken together, microbial cfDNA sequencing provides a novel opportunity allowing us to further understand the involvement of bloodborne microbes in development of diseases. Similar approaches should also be applicable to the study of metagenomics for sufficient and comprehensive analysis of microbial species living in various environments. This article reviews this line of research and discuss the methodological approaches that have been developed, or are likely to be developed in the future, which may have strong potential to facilitate cfDNA- and cfRNA-based studies of cancer and acute/chronic diseases, in the hope that a better understanding of the hidden microbes in the circulatory system will improve diagnosis, prevention and treatment of problematic diseases.

Efficient and unique cobarcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novo assembly.

Here, we describe single-tube long fragment read (stLFR), a technology that enables sequencing of data from long DNA molecules using economical second-generation sequencing technology. It is based on adding the same barcode sequence to subfragments of the original long DNA molecule (DNA cobarcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process, up to 3.6 billion unique barcode sequences were generated on beads, enabling practically nonredundant cobarcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique cobarcoding of more than 8 million 20- to 300-kb genomic DNA fragments. Analysis of the human genome NA12878 with stLFR demonstrated high-quality variant calling and phase block lengths up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries, and their construction did not significantly add to the time or cost of whole-genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high-quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

Nanometre electron beam sculpting of suspended graphene and hexagonal boron nitride heterostructures

Nano-patterned and suspended graphene membranes find applications in electronic devices, filtration and nano-pore DNA sequencing. However, the fabrication of suspended graphene structures with nanoscale features is challenging. We report the direct patterning of suspended membranes consisting of a graphene layer on top of a thin layer of hexagonal boron nitride which acts as a mechanical support, using a highly focused electron beam to fabricate structures with extremely high spatial resolution within the scanning transmission electron microscope (STEM). The boron nitride support enables the fabrication of stable graphene geometries by preventing intrinsic strain in graphene membranes from distorting the patterned features. Line cuts with widths below 2 nm are reported. We also demonstrate that the extent of cutting can be monitored in situ utilising electron energy loss spectroscopy (EELS).

Continuous Embeddings of DNA Sequencing Reads and Application to Metagenomics.

We propose a new model for fast classification of DNA sequences output by next-generation sequencing machines. The model, which we call fastDNA, embeds DNA sequences in a vector space by learning continuous low-dimensional representations of the k -mers it contains. We show on metagenomics benchmarks that it outperforms the state-of-the-art methods in terms of accuracy and scalability.

Validation of SSR Molecular Markers Linked to Drought Tolerant in Some Wheat Cultivars

The present study was carried out to conduct drought tolerance in three wheat cultivars including susceptible (Gemmiza7) and tolerant (Sakha93 and Sahel1). Molecular characterization was done by 26 SSR markers located on chromosome7 which was associated with drought tolerance in many previous studies. 26 SSR markers were polymorphic and thus showed 100% polymorphism. The number of alleles per locus varied from 2 to 3 alleles with an average (2.62). The polymorphism information content (PIC) value ranged from 0.34 to 0.59, with a mean of 0.51. The discrimination power (Dp) value ranged between 0.67 and 0.78 with an average of 0.71 per locus and Heterozygosity (He) value varied from 0.44 to 0.67 with an average of 0.59. The genetic relationships estimated by the polymorphism of SSR markers revealed a greater level of genetic variability in wheat cultivars of wide adaptability and applicability. Whereas an average of combined probability value for the SSR markers was 6.15 x 10-16, suggests the capability of the marker system to distinguish identity and purity of wheat cultivars. In addition to the SSR markers revealed various bands that were either absent or present within tolerant cultivars (Sakha93 and Sahel1) which were altogether absent in susceptible cultivar (Gemmiza7). Also, SSRs of diagnostic and curatorial importance were discerned as ‘stand-alone’ molecular descriptors for barcoding the application of DNA sequences of standardized genetic markers for the identification of wheat cultivars. However, the genetic information in this study could provide useful information to address breeding programs and germplasm resource management.

Multicenter experience with large panel next-generation sequencing in patients with advanced solid cancers in Japan.

Application of next-generation DNA sequencing (NGS) has recently become increasingly common in the field of clinical oncology in several countries around the world. In Japan also, a system for applying NGS to routine clinical practice is gradually being established. During this process, we introduced in Japan the tumor-profiling MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) assay. We present here our initial experience with the use of MSK-IMPACT in 68 patients selected from two institutions in Japan between June 2016 and October 2017. MSK-IMPACT sequencing was successful and yielded results in specimens obtained from 64 of the 68 patients, representing an overall assay success rate of 94.1%. The top three cancer types tested were endometrial cancer (17.2%), pancreatic cancer (15.6%) and colorectal cancer (12.5%). Evaluation of the clinical actionability of the genetic alterations revealed that 25.0% of patients (n = 16) harbored at least one actionable alteration. However, enrolling the patients in a genomically matched clinical trial was difficult, mainly because most clinical trials are limited to tumors arising from a specific organ/site. One patient with microsatellite instability-high status, as determined by MSK-IMPACT, was treated with pembrolizumab and showed partial response. Although tumor profiling by NGS and administration of genomically matched therapy is a promising strategy, because of its high cost, we need to consider how we can fit it into the Japanese medical system. Towards this end, we believe that it is important to share our initial experience for furthering precision medicine in Japan.

Adsorption characteristics of epigenetically modified DNA nucleobases on single-layer MoS2: A first-principles study

We report a comprehensive van der Waals density functional theory study on the adsorption characteristics of four canonical and six epigenetically modified DNA nucleobases on single-layer molybdenum disulfide (SL-MoS2) substrates. All the considered nucleobases are physisorbed on SL-MoS2 with a modest electron transfer, ranging from 0.007e− to 0.041e−, from the adsorbates to SL-MoS2. Upon the physisorption of adenine, cytosine, guanine, and thymine on the SL-MoS2 sheet, the work function is decreased by 0.29, 0.09, 0.54, and 0.01 eV, respectively. It is shown that the bandgap of SL-MoS2 can be significantly reduced, by as much as 30%, through the adsorption of nucleobase molecules as the induced molecular flat bands tend to appear either in the energy gap region or in the vicinity of the valence band maximum. We unfold the emergence of distinct electron energy loss spectra upon nucleobase adsorption which may serve as potential probes for selective detection of nucleobase molecules in prospective DNA sequencing applications based on SL-MoS2.

Taxa: An R package implementing data standards and methods for taxonomic data.

Thetaxa R package provides a set of tools for defining and manipulating taxonomic data. The recent and widespread application of DNA sequencing to community composition studies is making large data sets with taxonomic information commonplace. However, compared to typical tabular data, this information is encoded in many different ways and the hierarchical nature of taxonomic classifications makes it difficult to work with. There are many R packages that use taxonomic data to varying degrees but there is currently no cross-package standard for how this information is encoded and manipulated. We developed the R packagetaxa to provide a robust and flexible solution to storing and manipulating taxonomic data in R and any application-specific information associated with it.Taxa provides parsers that can read common sources of taxonomic information (taxon IDs, sequence IDs, taxon names, and classifications) from nearly any format while preserving associated data. Once parsed, the taxonomic data and any associated data can be manipulated using a cohesive set of functions modeled after the popular R packagedplyr. These functions take into account the hierarchical nature of taxa and can modify the taxonomy or associated data in such a way that both are kept in sync.Taxa is currently being used by themetacoder andtaxize packages, which provide broadly useful functionality that we hope will speed adoption by users and developers.

Transposase subunit architecture and its relationship to genome size and the rate of transposition in prokaryotes and eukaryotes.

Cut-and-paste transposons are important tools for mutagenesis, gene-delivery and DNA sequencing applications. At the molecular level, the most thoroughly understood are Tn5 and Tn10 in bacteria, and mariner and hAT elements in eukaryotes. All bacterial cut-and-paste transposases characterized to date are monomeric prior to interacting with the transposon end, while all eukaryotic transposases are multimers. Although there is a limited sample size, we proposed that this defines two pathways for transpososome assembly which distinguishes the mechanism of the bacterial and eukaryotic transposons. We predicted that the respective pathways would dictate how the rate of transposition is related to transposase concentration and genome size. Here, we have tested these predictions by creating a single-chain dimer version of the bacterial Tn5 transposase. We show that artificial dimerization switches the transpososome assembly pathway from the bacterial-style to the eukaryotic-style. Although this had no effect in vitro, where the transposase does not have to search far to locate the transposon ends, it increased the rate of transposition in bacterial and HeLa cell assays. However, in contrast to the mariner elements, the Tn5 single-chain dimer remained unaffected by over-production inhibition, which is an emergent property of the transposase subunit structure in the mariner elements.

Taxa: An R package implementing data standards and methods for taxonomic data

The taxa R package provides a set of tools for defining and manipulating taxonomic data. The recent and widespread application of DNA sequencing to community composition studies is making large data sets with taxonomic information commonplace. However, compared to typical tabular data, this information is encoded in many different ways and the hierarchical nature of taxonomic classifications makes it difficult to work with. There are many R packages that use taxonomic data to varying degrees but there is currently no cross-package standard for how this information is encoded and manipulated. We developed the R package taxa to provide a robust and flexible solution to storing and manipulating taxonomic data in R and any application-specific information associated with it. Taxa provides parsers that can read common sources of taxonomic information (taxon IDs, sequence IDs, taxon names, and classifications) from nearly any format while preserving associated data. Once parsed, the taxonomic data and any associated data can be manipulated using a cohesive set of functions modeled after the popular R package dplyr. These functions take into account the hierarchical nature of taxa and can modify the taxonomy or associated data in such a way that both are kept in sync. Taxa is currently being used by the metacoder and taxize packages, which provide broadly useful functionality that we hope will speed adoption by users and developers.

Fabrication of Defective Single Layers of Hexagonal Boron Nitride on Various Supports for Potential Applications in Catalysis and DNA Sequencing

Two-dimensional hexagonal boron nitride (hBN) has been shown to be a suitable substrate and gate material for high-performance graphene electronics and has been proposed for applications in catalysis and DNA sequencing. This study explores how highly charged ions can be used for defect engineering, i.e., to locally induce modifications in single layers of hBN. For this, we irradiated single layers of hBN on SiO2, Mo foil, and Ir(111) with highly charged Xeq+ ions of different charge states (up to q = 40) at a fixed kinetic energy of 260 keV. The ion-induced nanoscaled modifications are analyzed as a function of the charge state using atomic force microscopy in the friction force mode and secondary-ion mass spectrometry. The data show that a charge state of more than q = 28 corresponding to a minimum potential energy of 15–17 keV is sufficient to achieve defect creation via electronic excitation. This is higher in comparison to graphene on SiO2 (≃12 keV), which opens up the possibility of selective defect ...

Abstract 5348: Single-cell analysis of mutational heterogeneity in acute myeloid leukemia tumors with high-throughput droplet microfluidics

Abstract To make possible the routine characterization of genetic diversity within cancer cell populations, we developed a novel two-step microfluidic droplet workflow that enables efficient and massively-parallel single-cell PCR-based genomic barcoding for single-cell DNA sequencing applications. We demonstrate that the two-step microfluidic approach is required for robust DNA amplification on thousands of individual cells per run with high coverage uniformity and low allelic dropout of targeted genomic loci. To apply our single-cell sequencing technology to human tumor samples, we developed a targeted panel to partially sequence 26 genes frequently mutated in acute myeloid leukemia (AML) including TP53, DNMT3A, FLT3, NPM1, NRAS, IDH1 and IDH2. Using this panel, we were able to sensitively identify SNV and indel-defined clones within AML samples and assess their distribution at the time of diagnosis, remission and relapse. We also used single cell SNVs to monitor host and donor cell populations during bone marrow transplantation (BMT), which allowed us to accurately evaluate engraftment and disease relapse. Collectively, our single-cell data indicates that clonal populations inferred from VAFs obtained from bulk sequencing data may not fully resolve the heterogeneity within tumors; moreover, the single-cell nature of our approach enabled the unambiguous identification of multiple co-occurring mutations within subclones that is not possible with bulk measurements. Collectively, our results show a greater degree of heterogeneity in AML tumor samples than is commonly appreciated with traditional sequencing paradigms and demonstrate the value of single-cell analysis for AML. Citation Format: Dennis J. Eastburn, Maurizio Pellegrino, Adam Sciambi, Sebastian Treusch, Liwen Xu, Robert Durruthy-Durruthy, Kaustubh Gokhale, Jose Jacob, Tina X. Chen, William Oldham, Jairo Matthews, Hagop Kantarjian, Andrew Futreal, Keyur Patel, Keith W. Jones, James L. Zehnder, Koichi Takahashi. Single-cell analysis of mutational heterogeneity in acute myeloid leukemia tumors with high-throughput droplet microfluidics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5348.

Design of tunable-size 2D nanopore membranes from self-assembled amphiphilic nanosheets using dissipative particle dynamics simulations

Two-dimensional (2D) nanopore membranes play a pivotal role in the application of molecular separation, DNA sequencing, drug release and biosensing. Owing to their special properties including 2D symmetry and interfacial activity, amphiphilic nanosheets (ANs) have great potential for generating functional nanomaterials. In this work, the dissipative particle dynamics (DPD) simulations are employed to design 2D nanopore membranes with tunable size using AN molecules. We find that these two-dimensional membranes are much stable due to the double-layer assembled framework of the membrane with the thickness from 2.5 to 7.8 nm. Different sized nanopores ranging from 2.45 to 6.15 nm are also identified in the membrane architecture, which can be controlled by the concentration ratio, the length of grafting polymer and the affinity of ANs. Moreover, when at low concentration ratio (<10%), the ANs tend to form different core-shell structured micelles, including spherical, ellipsoidal and long rod-like self-assembled morphologies. From our study findings, the ANs could be extendedly used for drug delivery and release, multi-layer 2D nanomaterials by the advantage of their chemical tunability, including the affinity and stimulus-responsive properties of grafting polymer as well as the geometry of scaffold structure in the ANs.

Sequencing of the Cheese Microbiome and Its Relevance to Industry.

The microbiota of cheese plays a key role in determining its organoleptic and other physico-chemical properties. It is essential to understand the various contributions, positive or negative, of these microbial components in order to promote the growth of desirable taxa and, thus, characteristics. The recent application of high throughput DNA sequencing (HTS) facilitates an even more accurate identification of these microbes, and their functional properties, and has the potential to reveal those microbes, and associated pathways, responsible for favorable or unfavorable characteristics. This technology also facilitates a detailed analysis of the composition and functional potential of the microbiota of milk, curd, whey, mixed starters, processing environments, and how these contribute to the final cheese microbiota, and associated characteristics. Ultimately, this information can be harnessed by producers to optimize the quality, safety, and commercial value of their products. In this review we highlight a number of key studies in which HTS was employed to study the cheese microbiota, and pay particular attention to those of greatest relevance to industry.

Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples.

New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. Due to primer bias, the ability of metabarcoding to accurately depict biomass of different taxa from bulk communities remains unclear, while PCR-free whole mitochondrial genome (mitogenome) sequencing may provide a more reliable alternative. Here, we used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (three different amplicons) and shotgun mitogenome sequencing. Additionally, we used individual COI barcoding and de novo mitochondrial genome sequencing, to provide reference sequences for OTU assignment and metagenome mapping (mitogenome skimming), respectively. We found that, even though both methods occasionally failed to recover very low abundance species, metabarcoding was less consistent, by failing to recover some species with higher abundances, probably due to primer bias. Shotgun sequencing results provided highly significant correlations between read number and biomass in all but one species. Conversely, the read-biomass relationships obtained from metabarcoding varied across amplicons. Specifically, we found significant relationships for eight of 13 (amplicons B1FR-450bp, FF130R-130bp) or four of 13 (amplicon FFFR, 658bp) species. Combining the results of all three COI amplicons (multiamplicon approach) improved the read-biomass correlations for some of the species. Overall, mitogenomic sequencing yielded more informative predictions of biomass content from bulk macroinvertebrate communities than metabarcoding. However, for large-scale ecological studies, metabarcoding currently remains the most commonly used approach for diversity assessment.

Quantitative Evaluation of Dielectric Breakdown of Silicon Micro- and Nanofluidic Devices for Electrophoretic Transport of a Single DNA Molecule.

In the present study, we quantitatively evaluated dielectric breakdown in silicon-based micro- and nanofluidic devices under practical electrophoretic conditions by changing the thickness of the insulating layer. At higher buffer concentration, a silicon nanofluidic device with a 100 nm or 250 nm silicon dioxide layer tolerated dielectric breakdown up to ca. 10 V/cm, thereby allowing successful electrophoretic migration of a single DNA molecule through a nanochannel. The observed DNA migration behavior suggested that parameters, such as thickness of the insulating layer, tolerance of dielectric breakdown, and bonding status of silicon and glass substrate, should be optimized to achieve successful electrophoretic transport of a DNA molecule through a nanopore for nanopore-based DNA sequencing applications.

388 (PB-021) - Clinical application of tumour dna sequencing & tumour gene expression profiling in advanced metastatic breast cancer: A review of 36 patients tested between 2011 and 2017

388 (PB-021) - Clinical application of tumour dna sequencing & tumour gene expression profiling in advanced metastatic breast cancer: A review of 36 patients tested between 2011 and 2017

Taxa: An R package implementing data standards and methods for taxonomic data.

Thetaxa R package provides a set of tools for defining and manipulating taxonomic data. The recent and widespread application of DNA sequencing to community composition studies is making large data sets with taxonomic information commonplace. However, compared to typical tabular data, this information is encoded in many different ways and the hierarchical nature of taxonomic classifications makes it difficult to work with. There are many R packages that use taxonomic data to varying degrees but there is currently no cross-package standard for how this information is encoded and manipulated. We developed the R packagetaxa to provide a robust and flexible solution to storing and manipulating taxonomic data in R and any application-specific information associated with it.Taxa provides parsers that can read common sources of taxonomic information (taxon IDs, sequence IDs, taxon names, and classifications) from nearly any format while preserving associated data. Once parsed, the taxonomic data and any associated data can be manipulated using a cohesive set of functions modeled after the popular R packagedplyr. These functions take into account the hierarchical nature of taxa and can modify the taxonomy or associated data in such a way that both are kept in sync.Taxa is currently being used by themetacoder andtaxize packages, which provide broadly useful functionality that we hope will speed adoption by users and developers.

A Novel Biological Nanopore for Active DNA Transport and Detection

As reported, scientists have found many kinds of biological pores to study the stochastic sensing. Nanopore sensing has been developed as a platform technology and those biological porins have been reengineered with enormous probability for single-molecule DNA/RNA sequencing, such as α-Hemolysin channel and mycobacterial porin. Here we report a novel porin having a narrow diameter of 1.3-nm with a transmembrane region. This porin can be reconstructed into lipid bilayer and transport ions, small molecules, DNA and RNA. Interestingly, we find that both single-stranded and double-stranded nucleic acids can be translocated. The average dwell time for 48-nt ssDNA was 2-4 mS, and the blockade was 80%±5%. We also find its DNA helicase activity fueled by ATP hydrolysis. This nanopore has the potential application in active DNA transport and sequencing.

Nucleobases-decorated boron nitride nanoribbons for electrochemical biosensing: a dispersion-corrected DFT study.

Understanding the interactions between biomolecules and boron nitride nanostructures is key for their use in nanobiotechnology and medical engineering. In this study, we investigated the adsorption of nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) over armchair and zigzag boron nitride nanoribbons (BNNR) using density functional theory to define the applicability of BNNR for the sensing of nucleobases and DNA sequencing. To appropriately account for dispersion, the van der Waals forces (DFT-D2)-type method developed by Grimme was also included in the calculations. The calculated adsorption energy suggests the following order of adsorption for A-BNNR and Z-BNNR with the nucleobases: G > T > A > U > C and G > C > A > T > U, respectively. The origin of the binding of the different nucleobases with BNNR was analysed and π-π stacking was found to be responsible. In addition, the electronic properties, density of states and work function significantly vary after adsorption. These analyses indicate different binding natures for different nucleobases and BNNRs. Thus, this study demonstrates that BNNR can be applied as biosensors for the detection of nucleobases, which are constituents of DNA and RNA. Furthermore, analysis of electronic properties and adsorption energies will play a key role in targeted drug delivery, enzyme activities and genome sequencing. Our results indicate that BNNRs have better adsorption capacity than graphene and boron nitride nanotubes.