DNA Sequencing Applications Research Articles

Characterization of Bacterial Microbiota of P.D.O. Feta Cheese by 16S Metagenomic Analysis.

Background: The identification of bacterial species in fermented PDO (protected designation of origin) cheese is important since they contribute significantly to the final organoleptic properties, the ripening process, the shelf life, the safety and the overall quality of cheese. Methods: Ten commercial PDO feta cheeses from two geographic regions of Greece, Epirus and Thessaly, were analyzed by 16S metagenomic analysis. Results: The biodiversity of all the tested feta cheese samples consisted of five phyla, 17 families, 38 genera and 59 bacterial species. The dominant phylum identified was Firmicutes (49% of the species), followed by Proteobacteria (39% of the species), Bacteroidetes (7% of the species), Actinobacteria (4% of the species) and Tenericutes (1% of the species). Streptococcaceae and Lactobacillaceae were the most abundant families, in which starter cultures of lactic acid bacteria (LAB) belonged, but also 21 nonstarter lactic acid bacteria (NSLAB) were identified. Both geographical areas showed a distinctive microbiota fingerprint, which was ultimately overlapped by the application of starter cultures. In the rare biosphere of the feta cheese, Zobellella taiwanensis and Vibrio diazotrophicus, two Gram-negative bacteria which were not previously reported in dairy samples, were identified. Conclusions: The application of high-throughput DNA sequencing may provide a detailed microbial profile of commercial feta cheese produced with pasteurized milk.

Application of Restriction Site-Associated DNA Sequencing (RAD-Seq) for Copy Number Variation and Triploidy Detection in Human

At present, low-pass whole-genome sequencing (WGS) is frequently used in clinical research and in the screening of copy number variations (CNVs). However, there are still some challenges in the detection of triploids. Restriction site-associated DNA sequencing (RAD-Seq) technology is a reduced-representation genome sequencing technology developed based on next-generation sequencing. Here, we verified whether RAD-Seq could be employed to detect CNVs and triploids. In this study, genomic DNA of 11 samples was extracted employing a routine method and used to build libraries. Five cell lines of known karyotypes and 6 triploid abortion tissue samples were included for RAD-Seq testing. The triploid samples were confirmed by STR analysis and also tested by low-pass WGS. The accuracy and efficiency of detecting CNVs and triploids by RAD-Seq were then assessed, compared with low-pass WGS. In our results, RAD-Seq detected 11 out of 11 (100%) chromosomal abnormalities, including 4 deletions and 1 aneuploidy in the purchased cell lines and all triploid samples. By contrast, these triploids were missed by low-pass WGS. Furthermore, RAD-Seq showed a higher resolution and more accurate allele frequency in the detection of triploids than low-pass WGS. Our study shows that, compared with low-pass WGS, RAD-Seq has relatively higher accuracy in CNV detection at a similar cost and is capable of identifying triploids. Therefore, the application of this technique in medical genetics has a significant potential value.

Application of Massive Parallel DNA Sequencing to Diagnose Drug-Resistant Epilepsy in Children

Application of Massive Parallel DNA Sequencing to Diagnose Drug-Resistant Epilepsy in Children

Applications of Single-Cell DNA Sequencing.

Over the past decade, genomic analyses of single cells-the fundamental units of life-have become possible. Single-cell DNA sequencing has shed light on biological questions that were previously inaccessible across diverse fields of research, including somatic mutagenesis, organismal development, genome function, and microbiology. Single-cell DNA sequencing also promises significant future biomedical and clinical impact, spanning oncology, fertility, and beyond. While single-cell approaches that profile RNA and protein have greatly expanded our understanding of cellular diversity, many fundamental questions in biology and important biomedical applications require analysis of the DNA of single cells. Here, we review the applications and biological questions for which single-cell DNA sequencing is uniquely suited or required. We include a discussion of the fields that will be impacted by single-cell DNA sequencing as the technology continues to advance.

Computer vision AC-STEM automated image analysis for 2D nanopore applications

Transmission electron microscopy (TEM) has led to important discoveries in atomic imaging and as an atom-by-atom fabrication tool. Using electron beams, atomic structures can be patterned, annealed and crystallized, and nanopores can be drilled in thin membranes. We review current progress in TEM analysis and implement a computer vision nanopore-detection algorithm that achieves a 96% pixelwise precision in TEM images of nanopores in 2D membranes (WS2), and discuss parameter optimization including a variation on the traditional grid search and gradient ascent. Such nanopores have applications in ion detection, water filtration, and DNA sequencing, where ionic conductance through the pore should be concordant with its TEM-measured size. Standard computer vision methods have their advantages as they are intuitive and do not require extensive training data. For completeness, we briefly comment on related machine learning for 2D materials analysis and discuss relevant progress in these fields. Image analysis alongside TEM allows correlated fabrication and analysis done simultaneously in situ to engineer devices at the atomic scale.

Genetic Disease and Therapy.

Genetic diseases cause numerous complex and intractable pathologies. DNA sequences encoding each human's complexity and many disease risks are contained in the mitochondrial genome, nuclear genome, and microbial metagenome. Diagnosis of these diseases has unified around applications of next-generation DNA sequencing. However, translating specific genetic diagnoses into targeted genetic therapies remains a central goal. To date, genetic therapies have fallen into three broad categories: bulk replacement of affected genetic compartments with a new exogenous genome, nontargeted addition of exogenous genetic material to compensate for genetic errors, and most recently, direct correction of causative genetic alterations using gene editing. Generalized methods of diagnosis, therapy, and reagent delivery into each genetic compartment will accelerate the next generations of curative genetic therapies. We discuss the structure and variability of the mitochondrial, nuclear, and microbial metagenomic compartments, as well as the historical development and current practice of genetic diagnostics and gene therapies targeting each compartment.

Distribution and therapeutic outcomes of intergenic sequence-ALK fusion and coexisting ALK fusions in lung adenocarcinoma patients

IntroductionPatients with ALK rearranged non-small-cell lung cancer (NSCLC) show survival benefits from tyrosine-kinase inhibitor (TKI). Widely application of DNA sequencing revealed various rearrangement pattern in addition to single EML4-ALK fusion. Here, we retrospectively analyzed the distribution and coexistence of ALK rearrangement and therapeutic outcome of patients with ALK rearranged NSCLC. MethodALK positive NSCLC patients were screened at West China Hospital. NGS was performed on pre-treatment samples. Clinical characteristics and therapeutic outcomes were collected to retrospectively analyzed. ResultsAmong the 89 patients with 22 ALK rearrangements, fusions of intergenic sequences with ALK were found in 15 (16.85 %). Non-EML4–ALK fusions were present in 18 patients (20.22 %). Coexistence of rearrangements were present in 16 patients (17.98 %). Intergenic sequence–ALK and non-EML4–ALK fusions occurred at higher rates in patients with at least two fusions (62.5 % versus 6.85 % for intergenic sequence–ALK, 62.5 % versus 10.96 % for non-EML4–ALK). There were 40 ALK-rearranged NSCLC patients receiving the first-line crizotinib. The median progression-free survival (PFS) was 9.7 months when excluding three lost patients. In the seven patients who had at least two fusions, the median PFS was 11.9 months, compared with 9.0 months among those with single (p = 0.336). No significant difference in median PFS was found between patients with and without intergenic-ALK fusion (12.0 months versus 9.6 months, p = 0.989). The median PFS was 9.0 months in patients harboring a single EML4-ALK fusion versus 13.0 months in those with other ALK alterations (P = 0.890). The PFS of patients with single intergenic sequence-ALK fusion reached to 2.9 months, 27 months, and 28.9 months respectively. ConclusionOur study reports the distribution of intergenic sequence–ALK and coexisting fusions in ALK-rearranged NSCLC. Intergenic sequence–ALK and non-EML4-ALK are prone to coexist with other fusions. Neither intergenic sequence–ALK nor coexistence of fusions had a significant effect on the therapeutic benefit of treatment with crizotinib.

Sequencing and Reconstructing Helminth Mitochondrial Genomes Directly from Genomic Next-Generation Sequencing Data.

We present a detailed method for extraction of high-molecular weight genomic DNA suitable for numerous DNA sequencing applications, and a straightforward in silico approach for reconstructing novel mitochondrial (mt) genomes directly from total genomic DNA extracts derived from next-generation sequencing (NGS) data sets. The in silico post-sequencing pipeline described is fast, accurate, and highly efficient, with modest memory requirements that can be performed using a standard desktop computer. The approach is particularly effective for obtaining mitochondrial genomes for species with little or no mitochondrial sequence information currently available and overcomes many of the limitations of traditional strategies. The described methodologies are also applicable for metagenomics sequencing from mixed or pooled samples containing multiple species and subsequent specific assembly of specific mitochondrial genomes.

CYCLIC PARTIAL WORDS

Partial words are linear words with holes. Cyclic words are derived from linear words by linking its first letter after the last one. Both partial words and cyclic words have wide applications in DNA sequencing. In this paper we introduce cyclic partial words and discuss their periodicity and certain properties. We also establish representation of a cyclic partial word using trees.

Preparation of Fragaceatoxin C (FraC) Nanopores.

Biological nanopores are an emerging class of biosensors with high-end precision owing to their reproducible fabrication at the nanometer scale. Most notably, nanopore-based DNA sequencing applications are currently being commercialized, while nanopore-based proteomics may become a reality in the near future.Although membrane proteins often prove to be difficult to purify, we describe a straightforward protocol for the preparation of Fragaceatoxin C (FraC) nanopores, which may have applications for DNA analysis and nanopore-based proteomics. Recombinantly expressed FraC nanopores are purified via two rounds of Ni-NTA affinity chromatography before and after oligomerization on sphingomyelin-containing liposomes. Starting from a plasmid vector containing the FraC gene, our method allows the production of purified nanopores within a week. Afterward, the FraC nanopores can be stored at +4°C for several months, or frozen.

Abstract 3120: Genetic heterogeneity profiling for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application of DEPArrayTM System and single cell DNA sequencing for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application DEPArrayTM system and single cell DNA sequencing

Abstract Introduction: Although CTCs display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. We previously reported that overexpression of HER2 in CTCs is associated with worse prognosis in MBC (2018 AACR #5195). Herein, we report a new finding of heterogeneity profiling for HER2 positive CTC by using a new approach for single CTC isolation and sequencing. Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV MBC patients before therapy. CTC enumeration were performed in FDA approved CellSearch™ System by targeting the EpCAM antigen for capturing CTCs which were then stained by Anti-CK-PE, DAPI, anti-CD45-APC and anti-HER2-FITC. The single HER2+ CTCs were isolated by using DEPArrayTM System (Menarini). The single cell DNA was isolated and the initial library was prepared by SMARTer® PicoPLEX® Gold Single Cell DNA-Seq Kit, and the exome capture was performed by Twist Human Core Exome EF Multiplex Complete Kit. The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina). It was a paired end run, 75×75 bps run with dual indexing. Results: We identified 208 CTCs by CellSearch™ system, including 114 HER2+ CTCs and 4 CTC-clusters. Single HER2+ CTC was isolated and sequenced. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 362,015 counts (58.23%) for intron variants, 104,011 counts (16.73%) for intergenic variants, 56,678 counts (9.19%) for exon variants, 41,133 counts (6.67%) for downstream genes, 33,903 counts (5.45%) for upstream genes and others (3.73%). There were 44 genes (64 sites) variants were identified to have highest impact effect (≥20) on HER2+ CTC chromosome, among which TP53, PIK3CA, ESR1, PIK3R1 variants matched the most recent genomic characterization report for MBC tissue (Bertucci F. Nature 2019) when the XYLB, CYP17A1, CA2 and CAD variants were firstly reported. More important, although overexpression of PTP1B (PTPN1) was implicated in the development of MBC, our evidences firstly demonstrated that PTPN1 mutation is the most important variant (≥20) in CTCs for MBC. Furthermore, we also found that ESR1 mutation in CTC correlated with ESR1 status in ctDNA. Except for the high impact variants, there were 42 genes (66 sites) and 83 genes (136 sites) have medium (10-19) and significant (5-9) impact variants respectively. Conclusion: Genomic characterization of HER2 positive single CTCs will elucidate new specific gene alterations (e.g. PTPN1) associated with disease metastasis, which will improve understanding of this critical process in MBC and lead to the development of novel drugs aimed at their eradication using molecularly driven therapies for this deadly condition. Citation Format: Qiang Zhang, Lorenzo Gerratana, Kara L. Pivarski, Xinkun Wang, Zhe Ji, Emily Stroup, Elena Vagia Vagia, Andrew Adam Davis A. Adam Davis, Ami Shah, Katy L. Kerby, Lisa Flaum Flaum, Firas Wehbe, Youbin Zhang, Wenan Qiang, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Genetic heterogeneity profiling for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application of DEPArrayTM System and single cell DNA sequencing for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application DEPArrayTM system and single cell DNA sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3120.

Palaeomicrobiology: Application of Ancient DNA Sequencing to Better Understand Bacterial Genome Evolution and Adaptation

Next Generation Sequencing (NGS) has unlocked access to the wide range of non-cultivable microorganisms, including those present in the ancient past. The study of microorganisms from ancient sources (palaeomicrobiology) using DNA sequencing now provides a unique opportunity to examine ancient microbial genomic content, explore pathogenicity, and understand microbial evolution in greater detail than ever before. As a result, current studies have focused on reconstructing the evolutionary history of a number of human pathogens involved in ancient and historic pandemic events. These studies have opened the door for a variety of future palaeomicrobiology studies, which can focus on commensal microorganisms, species from non-human hosts, information from host-genomics, and the use of bacteria as proxies for additional information about past human health, behavior, migration, and culture. Here, we describe the origin and the historical and recent advances in the field of palaeomicrobiology, review some of the most notable ancient pathogenic microorganism studies, and provide perspectives on how NGS and whole genome information from ancient microorganisms contribute to our understanding of bacterial evolution on a broader scale. We conclude by exploring the application of newly developed tools in palaeomicrobiology and discussing how future studies can improve our current understanding of the co-evolution relationships between humans and non-pathogenic microbes.

Rediscovering an old foe: Optimised molecular methods for DNA extraction and sequencing applications for fungarium specimens of powdery mildew (Erysiphales)

The purpose of this study was to identify a reliable DNA extraction protocol to use on 25-year-old powdery mildew specimens from the reference collection VPRI in order to produce high quality sequences suitable to address taxonomic phylogenetic questions. We tested 13 extraction protocols and two library preparation kits and found the combination of the E.Z.N.A.® Forensic DNA kit for DNA extraction and the NuGen Ovation® Ultralow System library preparation kit was the most suitable for this purpose.

Crowding-Induced DNA Translocation through a Protein Nanopore.

A crowded cellular environment is highly associated with many significant biological processes. However, the effect of molecular crowding on the translocation behavior of DNA through a pore has not been explored. Here, we use nanopore single-molecule analytical technique to quantify the thermodynamics and kinetics of DNA transport under heterogeneous cosolute PEGs. The results demonstrate that the frequency of the translocation event exhibits a nonmonotonic dependence on the crowding agent size, while both the event frequency and translocation time increase monotonically with increasing crowder concentration. In the presence of PEGs, the rate of DNA capture into the nanopore elevates 118.27-fold, and at the same time the translocation velocity decreases from 20 to 120 μs/base. Interestingly, the impact of PEG 4k on the DNA-nanopore interaction is the most notable, with up to ΔΔG = 16.27 kJ mol-1 change in free energy and 764.50-fold increase in the binding constant at concentration of 40% (w/v). The molecular crowding effect will has broad applications in nanopore biosensing and nanopore DNA sequencing in which the strategy to capture analyte and to control the transport is urgently required.

Recent Progress in Solid-State Nanopore DNA Sequencing

Solid-state nanopores offer a novel platform for single molecule analysis and characterization. In particular, they have attracted significant attention as tools for high-throughput, robust, and low-error DNA sequencing. The successful implementation of solid-state nanopores in emerging third-generation DNA sequencing applications is contingent upon developing methods for scalable fabrication, high-accuracy output, and integration with low-noise electronic architectures. In this presentation, I will cover recent progress our group has made in the production, characterization, and integration of these devices. New high-resolution methods for fabricating nanopores down to the single-atom level (0.5 nm) - ion beam irradiation, photo-oxidation, and controlled chemical etching - will first be discussed. I will then describe recent successes in incorporating solid-state nanopores with high-frequency (10 MHz), low-noise amplifiers and subsequent advances in signal processing. Lastly, a brief perspective on the outlook of solid-state nanopores as well as ongoing work with ion-channel-like pores in two-dimensional (2D) materials will be provided.

ELECTRICAL NOISES REDUCTION IN NANOPORES EXPERIMENTS BASED ON CONSENSUS FILTER

Nanopore has developed into an indispensable tool for single molecules experiments, where useful information about the characteristics of DNA/RNA can be obtained from the flowing of ionic current elicited by individual molecules as they traverse a single nanometer-scale aperture. However, due to the relatively high background current noise and low measurement bandwidth, it is difficult to distinguish the molecule-specific signals from the intrinsic noises of the nanopore recording systems, which impeded its application in DNA sequencing. In this paper, we present a novel consensus filter-based noise reduction approach, which considerably improves the accuracy of the noise-filtered current traces and the signal-to-noise ratios (SNRs). The new approach is compared to the conventional Bessel filters and validated by analyzing simulated representative translocation data as well as alpha-Hemolysin (α-HL) nanopore experimental data. It is demonstrated that the high frequency noise generated by the voltage noise of the amplifier and Analog-to-Digital converter circuits (ADC) can be significantly reduced and some hidden short-lived translocation events can be recovered with satisfying accuracy beyond the conventional limitations of the Bessel filters.

Legume genomics and transcriptomics: From classic breeding to modern technologies.

Legumes are essential and play a significant role in maintaining food standards and augmenting physiochemical soil properties through the biological nitrogen fixation process. Biotic and abiotic factors are the main factors limiting legume production. Classical breeding methodologies have been explored extensively about the problem of truncated yield in legumes but have not succeeded at the desired rate. Conventional breeding improved legume genotypes but with more resources and time. Recently, the invention of next-generation sequencing (NGS) and high-throughput methods for genotyping have opened new avenues for research and developments in legume studies. During the last decade, genome sequencing for many legume crops documented. Sequencing and re-sequencing of important legume species have made structural variation and functional genomics conceivable. NGS and other molecular techniques such as the development of markers; genotyping; high density genetic linkage maps; quantitative trait loci (QTLs) identification, expressed sequence tags (ESTs), single nucleotide polymorphisms (SNPs); and transcription factors incorporated into existing breeding technologies have made possible the accurate and accelerated delivery of information for researchers. The application of genome sequencing, RNA sequencing (transcriptome sequencing), and DNA sequencing (re-sequencing) provide considerable insights for legume development and improvement programs.Moreover, RNA-Seq helps to characterize genes, including differentially expressed genes, and can be applied for functional genomics studies, especially when there is limited information available for the studied genomes. Genome-based crop development studies and the availability of genomics data as well as decision-making gears look be specific for breeding programs. This review mainly presents an overview of the path from classical breeding to new emerging genomics tools, which will trigger and accelerate genomics-assisted breeding for recognition of novel genes for yield and quality characters for sustainable legume crop production.

Shrinking nanopores shows potential for applications in DNA sequencing and detection

New analyses of shrinking nanopores reveals a method for obtaining control over nanopore size and thickness and understanding how the two affect each other.

Application of high-throughput DNA sequencing to score population-specific variants for rare disorders

Application of high-throughput DNA sequencing to score population-specific variants for rare disorders

Controlled size reduction and its underlying mechanism to form solid-state nanopores via electron beam induced carbon deposition

Solid-state nanopores have drawn considerable attention for their potential applications in DNA sequencing and nanoparticle analysis. However, fabrication of nanopores, especially those of diameter below 30 nm, requires sophisticated techniques. Here, a versatile method to controllably reduce the diameter of prefabricated large-size pores down to sub-30 nm without greatly increasing the effective pore depth from the original membrane thickness is shown. This method exploits carbon deposition achieved via hydrocarbon evaporation, induced by an incident beam of electrons, and subsequent dissociation of hydrocarbon to solid carbon deposits. The carbon deposition employs a conventional scanning electron microscope equipped with direct visual feedback, along with a stable hydrocarbon source nearby the sample. This work systematically studies how electron beam accelerating voltage, imaging magnification, initial pore size and membrane composition affect the process of pore size reduction. Secondary electrons generated in the membrane material are confirmed to be the main cause of the dissociation of hydrocarbon. Thicker carbon deposited on one side than on the other of the membrane results in an asymmetric nanopore shape and a rectifying ionic transport. A physico-phenomenological model combined with Monte Carlo simulations is proposed to account for the observed carbon deposition behaviors.