645 results on '"DNA nanoball sequencing"'
Search Results
2. BGISEQ-500RS sequencing of a 448-plex SNP panel for forensic individual identification and kinship analysis
- Author
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Shujin Li, Guan-Ju Ma, Chi Zhang, Le Wang, Guang-Bin Zhao, and Ke-Lai Kang
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Forensic Genetics ,DNA nanoball sequencing ,Genotype ,High-Throughput Nucleotide Sequencing ,Single-nucleotide polymorphism ,Computational biology ,Sequence Analysis, DNA ,Biology ,DNA Fingerprinting ,Polymorphism, Single Nucleotide ,DNA sequencing ,Deep sequencing ,Pathology and Forensic Medicine ,SNP genotyping ,genomic DNA ,Genetics ,SNP ,Humans ,Genotyping ,Microsatellite Repeats - Abstract
Next generation sequencing (NGS)-based single nucleotide polymorphism (SNP) genotyping is widely used in the field of forensics. SNP genotyping data from several NGS platforms have been published, but forensic application trials of DNA nanoball sequencing platforms have been very limited. In this work, we developed a 448-plex SNP panel on the BGISEQ-500RS platform. The sequencing metrics of a total of 261 samples that were sequenced with this panel are reported in detail. The average sequencing depth was 8373 × and the average heterozygosity of the 448-plex assay was 0.85. Sensitivity analysis showed that 325 SNPs were successfully genotyped with as little as 50 pg of genomic DNA, with the mean quality score of the sequencing data above Q30. Forensic parameters were calculated based on the data of 142 unrelated Chinese Han individuals and the combined matching probability was as low as 5.21 × 10−101. Kinship analyses based on experiments and computer simulations showed that the 448-panel was as effective as the ForenSeq™ DNA Signature Prep Kit for second-degree kinship identification, and when the two panels were merged, the related pairs were almost completely distinguished from unrelated pairs. The 448-plex SNP panel on the BGISEQ-500RS platform provides a powerful tool for forensic individual identification and kinship analysis.
- Published
- 2021
3. The architecture of SARS-CoV-2 transcriptome
- Author
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Joo Yeon Lee, V. Narry Kim, Jun Won Kim, Dong Wan Kim, Hyeshik Chang, and Jeong Sun Yang
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DNA nanoball sequencing ,Polyadenylation ,viruses ,Computational biology ,Biology ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Epigenesis, Genetic ,Frameshift mutation ,Transcriptome ,Betacoronavirus ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,Chlorocebus aethiops ,Animals ,RNA Processing, Post-Transcriptional ,ORFS ,Vero Cells ,030304 developmental biology ,Subgenomic mRNA ,0303 health sciences ,SARS-CoV-2 ,Sequence Analysis, RNA ,RNA ,biology.organism_classification ,RNA, Viral ,030217 neurology & neurosurgery - Abstract
SARS-CoV-2 is a betacoronavirus that is responsible for the COVID-19 pandemic. The genome of SARS-CoV-2 was reported recently, but its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we here present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic RNA and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces a large number of transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif being AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the internal modification and the 3′ tail. Functional investigation of the unknown ORFs and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.HighlightsWe provide a high-resolution map of SARS-CoV-2 transcriptome and epitranscriptome using nanopore direct RNA sequencing and DNA nanoball sequencing.The transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical.In addition to the genomic and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces transcripts encoding unknown ORFs.We discover at least 41 potential RNA modification sites with an AAGAA motif.
- Published
- 2020
4. Highly comparable metabarcoding results from MGI-Tech and Illumina sequencing platforms
- Author
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Vladimir S. Mikryukov, Kęstutis Armolaitis, Sten Anslan, Lars Vesterdal, Leho Tedersoo, Inger Kappel Schmidt, Jelena Ankuda, Dagnija Lazdina, and Kristaps Makovskis
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DNA nanoball sequencing ,Operational taxonomic unit ,Bioinformatics ,NovaSeq ,Sequencing data ,Statistical difference ,Computational biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,COI ,Illumina ,DNBSEQ ,Molecular Biology ,Illumina dye sequencing ,MGI-Tech ,General Neuroscience ,Coi gene ,Soil dna ,Genomics ,General Medicine ,Amplicon ,Metabarcoding ,Medicine ,General Agricultural and Biological Sciences ,Entomology ,Zoology - Abstract
With the developments in DNA nanoball sequencing technologies and the emergence of new platforms, there is an increasing interest in their performance in comparison with the widely used sequencing-by-synthesis methods. Here, we test the consistency of metabarcoding results from DNBSEQ-G400RS (DNA nanoball sequencing platform by MGI-Tech) and NovaSeq 6000 (sequencing-by-synthesis platform by Illumina) platforms using technical replicates of DNA libraries that consist of COI gene amplicons from 120 soil DNA samples. By subjecting raw sequencing data from both platforms to a uniform bioinformatics processing, we found that the proportion of high-quality reads passing through the filtering steps was similar in both datasets. Per-sample operational taxonomic unit (OTU) and amplicon sequence variant (ASV) richness patterns were highly correlated, but sequencing data from DNBSEQ-G400RS harbored a higher number of OTUs. This may be related to the lower dominance of most common OTUs in DNBSEQ data set (thus revealing higher richness by detecting rare taxa) and/or to a lower effective read quality leading to generation of spurious OTUs. However, there was no statistical difference in the ASV and post-clustered ASV richness between platforms, suggesting that additional denoising step in the ASV workflow had effectively removed the ‘noisy’ reads. Both OTU-based and ASV-based composition were strongly correlated between the sequencing platforms, with essentially interchangeable results. Therefore, we conclude that DNBSEQ-G400RS and NovaSeq 6000 are both equally efficient high-throughput sequencing platforms to be utilized in studies aiming to apply the metabarcoding approach, but the main benefit of the former is related to lower sequencing cost.
- Published
- 2021
5. Exome Sequencing and Advances in Crop Improvement.
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Singh, Devi, Singh, Pankaj K., Chaudhary, Sarika, Mehla, Kamiya, and Kumar, Shashi
- Abstract
Abstract: Next-generation sequencing strategies have opened new vistas for molecular plant breeding. The sequence information obtained by the advent of next-generation sequencing provides a valuable tool not only for improving domesticated crops but also for investigating the natural evolution of crops. Such information provides an enormous potential for sustainable agriculture. In this review, we discuss how such sequencing approaches have transformed exome sequencing into a practical utility that has enormous potential for crop improvement in agriculture. Furthermore, we also describe the future of crop improvement beyond the exome sequencing strategies. [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
- View/download PDF
6. Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations
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Qing Wang, Grace M. O’leary, Pheobe S. Tang, Xu Wang, and Ming Zhang
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0301 basic medicine ,DNA nanoball sequencing ,Science ,DNA Mutational Analysis ,Biology ,Barcode ,Genome ,DNA sequencing ,Article ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,law ,Neoplasms ,Humans ,A-DNA ,Exome sequencing ,Genetics ,Multidisciplinary ,Genome, Human ,High-Throughput Nucleotide Sequencing ,DNA ,RNA Probes ,Neoplasm Proteins ,030104 developmental biology ,chemistry ,Duplex (building) ,Mutation ,RNA ,Medicine - Abstract
Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq). This method combines an ultra-sensitive single-stranded library construction with barcoding error correction, termed DEEPER-Library; and a DNA capture approach using RNA probes targeting both DNA strands, termed DEEPER-Capture. DEEPER-Seq can create NGS libraries from as little as 20 pg DNA with PCR error correcting capabilities, and capture target sequences at an average ratio of 29.2% by targeting both DNA strands simultaneously with an over 98.6% coverage. Our method tags and sequences each of the two strands of a DNA duplex independently and only scores mutations that are found at the same position in both strands, which allows us to identify mutations with allelic fractions down to 0.03% in a whole exome sequencing (WES) study with a background error rate of one artificial error per 4.8 × 109 nucleotides.
- Published
- 2017
7. Sensitive detection of DNA methyltransferase using the dendritic rolling circle amplification-induced fluorescence
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Xiaoru Zhang, Yawen Luan, Peng He, Xiaoyan Guo, and Weiling Song
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DNA nanoball sequencing ,Deoxyribozyme ,010402 general chemistry ,01 natural sciences ,Biochemistry ,DNA methyltransferase ,Fluorescence ,Analytical Chemistry ,chemistry.chemical_compound ,Environmental Chemistry ,DNA Modification Methylases ,Spectroscopy ,Chemistry ,Hybridization probe ,010401 analytical chemistry ,DNA, Catalytic ,DNA Methylation ,0104 chemical sciences ,Restriction enzyme ,Rolling circle replication ,Nucleic Acid Conformation ,Primer (molecular biology) ,DNA Probes ,Nucleic Acid Amplification Techniques ,DNA - Abstract
The analysis of DNA methylation and MTase activities is very important in the early clinical diagnosis of cancer, on purposes of providing insights into the mechanism of gene repression and developing novel drugs of treating methylation-related diseases. Combining the dendritic rolling circle amplification and Mg 2+ -dependent DNAzyme with a function of catalyzing the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses, a new fluorescent method for DNA methyltransferase detection was reported. In the presence of DNA methyltransferases (MTase), the methylation-responsive sequence of double-stranded DNA probe was methylated and then cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hybrid DNA probe then functioned as a signal primer to initiate the dendritic rolling circle amplification reaction, containing a circular DNA and a structurally tailored hairpin structure. Subsequently, the circular nucleic acid template produced a complementary sequence to the Mg 2+ -dependent DNAzyme and a sequence identical to the loop region of the co-added hairpin structure. At last, a fluorescence readout signal was afforded by the DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate. This method enabled the analysis of the target MTase with a detection limit up to 0.36 U mL −1 , and a dynamic range was obtained from 1.0 to 10 U mL −1 . Moreover, the proposed strategy was successfully applied in real sample assay. With this assay, the inhibitors of MTase were evaluated and screened which might be helpful for the discovery of anticancer drugs.
- Published
- 2017
8. Targeted capture and sequencing of gene-sized DNA molecules
- Author
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Lawrence Percival-Alwyn, Ingo Hein, David Baker, Pirita Paajanen, Florian Jupe, Kamil Witek, Glenn J. Bryan, Jonathan D. G. Jones, Matthew D. Clark, Michael Giolai, and Walter Verweij
- Subjects
0106 biological sciences ,0301 basic medicine ,DNA nanoball sequencing ,Computational biology ,Biology ,Genes, Plant ,Solanum ,01 natural sciences ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Insert (molecular biology) ,03 medical and health sciences ,Exon ,Intergenic region ,Gene family ,Exome ,Gene ,Gene Library ,Models, Genetic ,High-Throughput Nucleotide Sequencing ,DNA ,Exons ,Genomics ,Sequence Analysis, DNA ,030104 developmental biology ,010606 plant biology & botany ,Biotechnology - Abstract
Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family—key genes in plant immune systems.
- Published
- 2016
9. Cell-free DNA
- Author
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Mark I. Evans, Hagit Shani, and Pe'er Dar
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0301 basic medicine ,education.field_of_study ,DNA nanoball sequencing ,030219 obstetrics & reproductive medicine ,Massive parallel sequencing ,Shotgun sequencing ,Biochemistry (medical) ,Clinical Biochemistry ,Population ,Biology ,Bioinformatics ,medicine.disease ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Cell-free fetal DNA ,Single cell sequencing ,medicine ,Trisomy ,education ,Exome sequencing - Abstract
Cell-free fetal DNA screening for Down syndrome has gained rapid acceptance over the past few years with increasing market penetration. Three main laboratory methodologies are currently used: a massive parallel shotgun sequencing (MPSS), a targeted massive parallel sequencing (t-MPS) and a single nucleotide polymorphism (SNP) based approach. Although each of these technologies has its own advantages and disadvantages, the performance of all was shown to be comparable and superior to that of traditional first-trimester screening for the detection of trisomy 21 in a routine prenatal population. Differences in performance were predominantly shown for chromosomal anomalies other than trisomy 21. Understanding the limitations and benefits of each technology is essential for proper counseling to patients. These technologies, as well as few investigational technologies described in this review, carry a great potential beyond screening for the common aneuploidies.
- Published
- 2016
10. Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing
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Toshikazu Bando, Gengo Kashiwazaki, Hiroshi Sugiyama, Junetha Syed, Shinsuke Sato, Rhys Dylan Taylor, Kaori Hashiya, and Anandhakumar Chandran
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0301 basic medicine ,Alkylating Agents ,DNA nanoball sequencing ,Alkylation ,Receptor, ErbB-2 ,Computational biology ,Biology ,Genome ,DNA sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,Chemical Biology and Nucleic Acid Chemistry ,Genetics ,Humans ,Pyrroles ,Promoter Regions, Genetic ,Base Sequence ,Genome, Human ,Oligonucleotide ,Imidazoles ,High-Throughput Nucleotide Sequencing ,DNA ,Sequencing by ligation ,Nylons ,genomic DNA ,030104 developmental biology ,chemistry ,Human genome ,lipids (amino acids, peptides, and proteins) - Abstract
Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2: showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing.
- Published
- 2016
11. Surface ligation-based resonance light scattering analysis of methylated genomic DNA on a microarray platform
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Zhen Lei, Zhenxin Wang, Dianjun Liu, Xia Liu, and Lan Ma
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0301 basic medicine ,DNA nanoball sequencing ,Metal Nanoparticles ,Biochemistry ,Analytical Chemistry ,03 medical and health sciences ,Cell Line, Tumor ,Electrochemistry ,Humans ,Scattering, Radiation ,Environmental Chemistry ,Methylated DNA immunoprecipitation ,Ligase chain reaction ,Spectroscopy ,chemistry.chemical_classification ,DNA ligase ,Multiple displacement amplification ,DNA ,Genomics ,DNA Methylation ,Molecular biology ,Sequencing by ligation ,030104 developmental biology ,chemistry ,Rolling circle replication ,DNA methylation ,Gold ,Nucleic Acid Amplification Techniques - Abstract
DNA methylation is a crucial epigenetic modification and is closely related to tumorigenesis. Herein, a surface ligation-based high throughput method combined with bisulfite treatment is developed for analysis of methylated genomic DNA. In this method, a DNA microarray is employed as a reaction platform, and resonance light scattering (RLS) of nanoparticles is used as the detection principle. The specificity stems from allele-specific ligation of Taq DNA ligase, which is further enhanced by improving the fidelity of Taq DNA ligase in a heterogeneous reaction. Two amplification techniques, rolling circle amplification (RCA) and silver enhancement, are employed after the ligation reaction and a gold nanoparticle (GNP) labeling procedure is used to amplify the signal. As little as 0.01% methylated DNA (i.e. 2 pmol L(-1)) can be distinguished from the cocktail of methylated and unmethylated DNA by the proposed method. More importantly, this method shows good accuracy and sensitivity in profiling the methylation level of genomic DNA of three selected colonic cancer cell lines. This strategy provides a high throughput alternative with reasonable sensitivity and resolution for cancer study and diagnosis.
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- 2016
12. The design and analysis of transposon insertion sequencing experiments
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Michael C. Chao, Matthew K. Waldor, Sören Abel, and Brigid M. Davis
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0301 basic medicine ,Transposable element ,Genetics ,Cancer genome sequencing ,DNA nanoball sequencing ,General Immunology and Microbiology ,Shotgun sequencing ,030106 microbiology ,Computational biology ,Biology ,Microbiology ,Article ,DNA sequencing ,Sequencing by ligation ,03 medical and health sciences ,030104 developmental biology ,Infectious Diseases ,Computational analysis ,Illumina dye sequencing - Abstract
Transposon insertion sequencing (TIS) is a powerful approach that can be extensively applied to the genome-wide definition of loci that are required for bacterial growth under diverse conditions. However, experimental design choices and stochastic biological processes can heavily influence the results of TIS experiments and affect downstream statistical analysis. In this Opinion article, we discuss TIS experimental parameters and how these factors relate to the benefits and limitations of the various statistical frameworks that can be applied to the computational analysis of TIS data.
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- 2016
- Full Text
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13. Erratum to: Whole Genome DNA Methylation Analysis of Brachypodium distachyon Using Next-Generation Sequencing (BS-seq)
- Author
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I-Hsuan Lin
- Subjects
0301 basic medicine ,Cancer genome sequencing ,Whole genome sequencing ,DNA nanoball sequencing ,biology ,Shotgun sequencing ,Computational biology ,biology.organism_classification ,03 medical and health sciences ,030104 developmental biology ,Illumina Methylation Assay ,Brachypodium distachyon ,Illumina dye sequencing ,Reference genome - Published
- 2018
14. The discrepancy among single nucleotide variants detected by DNA and RNA high throughput sequencing data
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Yan Guo, David C. Samuels, Yu Shyr, Shilin Zhao, and Quanhu Sheng
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0301 basic medicine ,Heterozygote ,RNA editing ,DNA nanoball sequencing ,Genotype ,lcsh:QH426-470 ,lcsh:Biotechnology ,Biology ,Polymorphism, Single Nucleotide ,Deep sequencing ,03 medical and health sciences ,0302 clinical medicine ,DNA-RNA difference ,lcsh:TP248.13-248.65 ,Genetics ,Illumina dye sequencing ,Exome sequencing ,Probability ,Whole genome sequencing ,Massive parallel sequencing ,Sequence Analysis, RNA ,Shotgun sequencing ,Research ,High-Throughput Nucleotide Sequencing ,Sequence Analysis, DNA ,Single nucleotide variant ,lcsh:Genetics ,030104 developmental biology ,Single cell sequencing ,030220 oncology & carcinogenesis ,Mutation ,Databases, Nucleic Acid ,Biotechnology - Abstract
Background High throughput sequencing technology enables the both the human genome and transcriptome to be screened at the single nucleotide resolution. Tools have been developed to infer single nucleotide variants (SNVs) from both DNA and RNA sequencing data. To evaluate how much difference can be expected between DNA and RNA sequencing data, and among tissue sources, we designed a study to examine the single nucleotide difference among five sources of high throughput sequencing data generated from the same individual, including exome sequencing from blood, tumor and adjacent normal tissue, and RNAseq from tumor and adjacent normal tissue. Results Through careful quality control and analysis of the SNVs, we found little difference between DNA-DNA pairs (1%–2%). However, between DNA-RNA pairs, SNV differences ranged anywhere from 10% to 20%. Conclusions Only a small portion of these differences can be explained by RNA editing. Instead, the majority of the DNA-RNA differences should be attributed to technical errors from sequencing and post-processing of RNAseq data. Our analysis results suggest that SNV detection using RNAseq is subject to high false positive rates. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4022-x) contains supplementary material, which is available to authorized users.
- Published
- 2017
15. DNA marker-assisted identification ofPrunusaccessions
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H. Nybom, J. Sehic, Stein Harald Hjeltnes, and Fuad Gaši
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Genetics ,Prunus ,chemistry.chemical_compound ,DNA nanoball sequencing ,chemistry ,Genetic marker ,Microsatellite ,Identification (biology) ,Horticulture ,Biology ,DNA ,Sequencing by ligation - Published
- 2015
16. High-fidelity target sequencing of individual molecules identified using barcode sequences:de novodetection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients
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Ryo Matoba, Takuya Hamakawa, Fumio Imamura, Kikuya Kato, Yoji Kukita, Junji Uchida, and Yuichiro Doki
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Male ,DNA nanoball sequencing ,Lung Neoplasms ,Plasma Cells ,Population ,Biology ,law.invention ,chemistry.chemical_compound ,Sequencing by hybridization ,Stomach Neoplasms ,law ,Neoplasms ,Genetics ,DNA Barcoding, Taxonomic ,Humans ,education ,Molecular Biology ,Polymerase chain reaction ,education.field_of_study ,massively parallel DNA sequencer ,circulating tumour DNA ,Multiple displacement amplification ,High-Throughput Nucleotide Sequencing ,Reproducibility of Results ,DNA, Neoplasm ,General Medicine ,Full Papers ,Molecular biology ,Sequencing by ligation ,DNA sequencer ,chemistry ,Mutation ,barcode sequences ,DNA - Abstract
Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA.
- Published
- 2015
17. Biosensing by Tandem Reactions of Structure Switching, Nucleolytic Digestion, and DNA Amplification of a DNA Assembly
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Wenqing Zhang, Yingfu Li, John D. Brennan, Qiang Zhang, and Meng Liu
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DNA nanoball sequencing ,biology ,Chemistry ,DNA polymerase ,Multiple displacement amplification ,Bacillus Phages ,Biosensing Techniques ,DNA ,DNA-Directed DNA Polymerase ,General Medicine ,General Chemistry ,Aptamers, Nucleotide ,Polymerase cycling assembly ,Molecular biology ,Catalysis ,Sequencing by ligation ,chemistry.chemical_compound ,Rolling circle replication ,Biophysics ,biology.protein ,DNA, Circular ,Nucleic Acid Amplification Techniques ,Single molecule real time sequencing - Abstract
ϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3'-5' digestion of single-stranded but not double-stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure-switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre-primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure-switching event that causes nucleolytic conversion of the pre-primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.
- Published
- 2015
18. Denoising DNA deep sequencing data—high-throughput sequencing errors and their correction
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David Laehnemann, Arndt Borkhardt, and Alice C. McHardy
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0301 basic medicine ,error profile ,error correction ,DNA nanoball sequencing ,bias ,Computer science ,Sequence assembly ,computer.software_genre ,DNA sequencing ,Deep sequencing ,03 medical and health sciences ,Software Review ,Humans ,Molecular Biology ,Illumina dye sequencing ,Polymorphism, Genetic ,Genome, Human ,DNA sequencing theory ,high-throughput sequencing ,Computational Biology ,High-Throughput Nucleotide Sequencing ,Ion semiconductor sequencing ,Genomics ,Sequence Analysis, DNA ,030104 developmental biology ,next-generation sequencing ,Nanopore sequencing ,Data mining ,computer ,Sequence Alignment ,error model ,Algorithms ,Software ,Information Systems - Abstract
Characterizing the errors generated by common high-throughput sequencing platforms and telling true genetic variation from technical artefacts are two interdependent steps, essential to many analyses such as single nucleotide variant calling, haplotype inference, sequence assembly and evolutionary studies. Both random and systematic errors can show a specific occurrence profile for each of the six prominent sequencing platforms surveyed here: 454 pyrosequencing, Complete Genomics DNA nanoball sequencing, Illumina sequencing by synthesis, Ion Torrent semiconductor sequencing, Pacific Biosciences single-molecule real-time sequencing and Oxford Nanopore sequencing. There is a large variety of programs available for error removal in sequencing read data, which differ in the error models and statistical techniques they use, the features of the data they analyse, the parameters they determine from them and the data structures and algorithms they use. We highlight the assumptions they make and for which data types these hold, providing guidance which tools to consider for benchmarking with regard to the data properties. While no benchmarking results are included here, such specific benchmarks would greatly inform tool choices and future software development. The development of stand-alone error correctors, as well as single nucleotide variant and haplotype callers, could also benefit from using more of the knowledge about error profiles and from (re)combining ideas from the existing approaches presented here.
- Published
- 2015
19. Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions
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Hanna Kim, Henry A. Erlich, and Cassandra Calloway
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Sanger sequencing ,Genetics ,Forensic Genetics ,DNA nanoball sequencing ,Massive parallel sequencing ,Forensic Science ,Shotgun sequencing ,High-Throughput Nucleotide Sequencing ,General Medicine ,Sequence Analysis, DNA ,Biology ,DNA, Mitochondrial ,Polymerase Chain Reaction ,DNA sequencing ,symbols.namesake ,Single cell sequencing ,symbols ,Humans ,Illumina dye sequencing ,Exome sequencing ,DNA Primers - Abstract
Aim To apply massively parallel and clonal sequencing (next generation sequencing or NGS) to the analysis of forensic mixed samples. Methods A duplex polymerase chain reaction (PCR) assay targeting the mitochondrial DNA (mtDNA) hypervariable regions I/II (HVI/HVII) was developed for NGS analysis on the Roche 454 GS Junior instrument. Eight sets of multiplex identifier-tagged 454 fusion primers were used in a combinatorial approach for amplification and deep sequencing of up to 64 samples in parallel. Results This assay was shown to be highly sensitive for sequencing limited DNA amounts ( ~ 100 mtDNA copies) and analyzing contrived and biological mixtures with low level variants ( ~ 1%) as well as “complex” mixtures (≥3 contributors). PCR artifact “hybrid” sequences generated by jumping PCR or template switching were observed at a low level (
- Published
- 2015
20. Template-directed ligation on repetitive DNA sequences: a chemical method to probe the length of Huntington DNA
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Anika Kern and Oliver Seitz
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congenital, hereditary, and neonatal diseases and abnormalities ,DNA nanoball sequencing ,Hybridization probe ,General Chemistry ,Biology ,Molecular biology ,Sequencing by ligation ,chemistry.chemical_compound ,genomic DNA ,Template ,chemistry ,Ligation ,Repeated sequence ,DNA - Abstract
Several genomic disorders are caused by an excessive number of DNA triplet repeats. We developed a DNA-templated reaction in which product formation occurs only when the number of repeats exceeds a threshold indicative for the outbreak of Chorea Huntington. The combined use of native chemical PNA ligation and auxiliary DNA probes enabled reactions on templates obtained from human genomic DNA.
- Published
- 2015
21. Isothermal rolling circle amplification of virus genomes for rapid antigen detection and typing
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Michael Brasino and Jennifer N. Cha
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DNA nanoball sequencing ,Phage display ,Phagemid ,Biosensing Techniques ,Genome, Viral ,Computational biology ,Biochemistry ,Genome ,Analytical Chemistry ,chemistry.chemical_compound ,Peptide Library ,Electrochemistry ,Animals ,Environmental Chemistry ,Bacteriophages ,Cloning, Molecular ,Spectroscopy ,biology ,Chemistry ,Temperature ,biology.organism_classification ,Molecular biology ,Restriction enzyme ,Filamentous bacteriophage ,Rolling circle replication ,Immunoglobulin G ,DNA, Viral ,Rabbits ,Genetic Engineering ,Nucleic Acid Amplification Techniques ,DNA - Abstract
In this work, isothermal rolling circle amplification (RCA) of the multi-kilobase genome of engineered filamentous bacteriophage is used to report the presence and identification of specific protein analytes in solution. First, bacteriophages were chosen as sensing platforms because peptides or antibodies that bind medically relevant targets can be isolated through phage display or expressed as fusions to their p3 and p8 coat proteins. Second, the circular, single-stranded genome contained within the phage serves as a natural large DNA template for a RCA reaction to rapidly generate exponential amounts of double stranded DNA in a single isothermal step that can be easily detected using low-cost fluorescent nucleic acid stains. Amplifying the entire phage genome also provides high detection sensitivities. Furthermore, since the sequence of the viral DNA can be easily modified with multiple restriction enzyme sites, a simple DNA digest can be applied to detect and identify multiple antigens simultaneously. The methods developed here will lead to protein sensors that are highly scalable to produce, can be run without complex biological equipment and do not require the use of multiple antibodies or high-cost fluorescent DNA probes or nucleotides.
- Published
- 2015
22. Ultrasensitive electrochemical detection of dual DNA targets based on G-quadruplex-mediated amplification
- Author
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Nandi Zhou, Tian Yaping, Shuling Wang, Yong Liu, and Xiaofan Sun
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chemistry.chemical_classification ,DNA ligase ,DNA nanoball sequencing ,Chemistry ,General Chemical Engineering ,General Chemistry ,G-quadruplex ,Molecular biology ,DNA sequencing ,chemistry.chemical_compound ,Biophysics ,Denaturation (biochemistry) ,Multiplex ,DNA ,Single molecule real time sequencing - Abstract
A novel strategy was established for simultaneous electrochemical detection of dual target DNAs. A template DNA was firstly designed to be complementary to target DNA 1, target DNA 2, and part of signal DNA, which contains a G-quadruplex forming sequence. In the presence of both target DNAs and signal DNA, these sequences hybridized with template DNA and were further ligated together to form a long strand under the catalysis of E. coli DNA ligase. After denaturation, the ligated sequence was dehybridized with template DNA and captured with a capture probe immobilized on the surface of a gold electrode. With the help of hemin, a G-quadrupelx–hemin complex can be formed on the surface of the gold electrode which produced a remarkable electrochemical signal. Therefore, the proposed DNA sensor was used to simultaneously detect dual target DNAs with extremely high sensitivity. For target DNA 1 and target DNA 2, detection limits of 100 fM and 7.4 fM were achieved respectively. The specificity of the sensor was verified by employing one-base mismatched and three-base mismatched sequences. In consideration of its potential for multiplex DNA sequence detection, the proposed DNA sensor may have great potential in the fields of microbial identification, disease diagnosis, food quality control and environmental monitoring.
- Published
- 2015
23. Fluorescence in situ hybridization with DNA probes derived from individual chromosomes and chromosome regions
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Nikolay B. Rubtsov, Anton Bogomolov, and Tatyana V. Karamysheva
- Subjects
Genetics ,DNA nanoball sequencing ,medicine.diagnostic_test ,Hybridization probe ,Biophysics ,Computational biology ,Biology ,Genome ,DNA sequencing ,Sequencing by hybridization ,Structural Biology ,Chromosome regions ,medicine ,Fluorescence in situ hybridization ,Comparative genomic hybridization - Abstract
A significant part of the eukaryotic genomes consists of repetitive DNA, which can form large clusters or distributed along euchromatic chromosome regions. Repeats located in chromosomal regions make a problem in analysis and identification of the chromosomal material with fluorescence in situ hybridization (FISH). In most cases, the identification of chromosome regions using FISH requires detection of the signal produced with unique sequences. The feasibility, advantages and disadvantages of traditional methods of suppression of repetitive DNA hybridization, methods of repeats-free probe construction and methods of chromosome-specific DNA sequences visualization using image processing of multicolor FISH results are considered in the paper. The efficiency of different techniques for DNA probe generation, different FISH protocols, and image processing of obtained microscopic images depends on the genomic size and structure of analyzing species. This problem was discussed and different approaches were considered for the analysis of the species with very large genome, rare species and species which specimens are too small in size to obtain the amount of genomic and Cot-1 DNA required for suppression of repetitive DNA hybridization.
- Published
- 2014
24. Single-cell template strand sequencing by Strand-seq enables the characterization of individual homologs
- Author
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Diana C.J. Spierings, Mark Hills, Ashley D. Sanders, Peter M. Lansdorp, Ester Falconer, Damage and Repair in Cancer Development and Cancer Treatment (DARE), and Stem Cell Aging Leukemia and Lymphoma (SALL)
- Subjects
0301 basic medicine ,DNA nanoball sequencing ,DNA, Single-Stranded ,Biology ,VARIANTS ,HUMAN GENOMES ,General Biochemistry, Genetics and Molecular Biology ,DNA sequencing ,03 medical and health sciences ,0302 clinical medicine ,Sequencing by hybridization ,Genomic library ,Alleles ,IN-VIVO ,COPY NUMBER VARIATION ,Genetics ,Staining and Labeling ,LANDSCAPE ,Shotgun sequencing ,FLOW CYTOMETRIC ANALYSIS ,PAIRED-END ,DNA replication ,REARRANGEMENTS ,Sequence Analysis, DNA ,Sequencing by ligation ,BROMODEOXYURIDINE ,STRUCTURAL VARIATION ,030104 developmental biology ,Coding strand ,Single-Cell Analysis ,030217 neurology & neurosurgery - Abstract
The ability to distinguish between genome sequences of homologous chromosomes in single cells is important for studies of copy-neutral genomic rearrangements (such as inversions and translocations), building chromosome-length haplotypes, refining genome assemblies, mapping sister chromatid exchange events and exploring cellular heterogeneity. Strand-seq is a single-cell sequencing technology that resolves the individual homologs within a cell by restricting sequence analysis to the DNA template strands used during DNA replication. This protocol, which takes up to 4 d to complete, relies on the directionality of DNA, in which each single strand of a DNA molecule is distinguished based on its 5'-3' orientation. Culturing cells in a thymidine analog for one round of cell division labels nascent DNA strands, allowing for their selective removal during genomic library construction. To preserve directionality of template strands, genomic preamplification is bypassed and labeled nascent strands are nicked and not amplified during library preparation. Each single-cell library is multiplexed for pooling and sequencing, and the resulting sequence data are aligned, mapping to either the minus or plus strand of the reference genome, to assign template strand states for each chromosome in the cell. The major adaptations to conventional single-cell sequencing protocols include harvesting of daughter cells after a single round of BrdU incorporation, bypassing of whole-genome amplification, and removal of the BrdU(+) strand during Strand-seq library preparation. By sequencing just template strands, the structure and identity of each homolog are preserved.
- Published
- 2017
25. Ultra-accurate Genome Sequencing and Haplotyping of Single Human Cells
- Author
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Peter Edge, Vineet Bafna, Xiaohua Huang, Kun Zhang, Hosuk Lee, Wai Keung Chu, and Vikas Bansal
- Subjects
Cancer genome sequencing ,Genetics ,0303 health sciences ,DNA nanoball sequencing ,Shotgun sequencing ,030305 genetics & heredity ,Sequence assembly ,Hybrid genome assembly ,Genome project ,Biology ,03 medical and health sciences ,Sequencing by hybridization ,Paired-end tag ,030304 developmental biology - Abstract
Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (Single-Stranded Sequencing using micrOfluidic Reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10−8 and average 500kb long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs.
- Published
- 2017
- Full Text
- View/download PDF
26. Single molecule sequencing of M13 virus genome without amplification
- Author
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Zhou Zhiliang, Huan Jin, Haomin Wu, Lidong Zeng, Pierre Ezanno, Zhao Luyang, Michael W. Deem, Yan Li, Yan Qin, Huan Shang, Deng Liwei, Jinsen Cai, Jiankui He, Wu Ping, Xu Weibin, Renli Zhang, Zheng Jiao, Huan Zhao, and Li Gailing
- Subjects
Genetics ,DNA nanoball sequencing ,DNA sequencer ,Massive parallel sequencing ,Shotgun sequencing ,Multiple displacement amplification ,Transmission electron microscopy DNA sequencing ,Biology ,Deep sequencing ,Single molecule real time sequencing - Abstract
Third generation sequencing is a direct measurement of DNA/RNA sequences at the single molecule level without amplification. In this study, we report sequencing of the genome of the M13 virus by a new single molecule sequencing platform. Our platform detects single molecule fluorescence by the total internal reflection microscope technique, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x and 100% coverage. The consensus sequence accuracy is 100%. We demonstrated that single molecule sequencing has no significant GC bias.
- Published
- 2017
- Full Text
- View/download PDF
27. Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq)
- Author
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Wolf Reik, Felix Krueger, Sébastien A. Smallwood, Stephen J. Clark, Heather J. Lee, and Gavin Kelsey
- Subjects
0301 basic medicine ,Cancer genome sequencing ,DNA nanoball sequencing ,Base Sequence ,Bisulfite sequencing ,Computational biology ,Genomics ,Sequence Analysis, DNA ,Biology ,DNA Methylation ,General Biochemistry, Genetics and Molecular Biology ,Bisulfite ,03 medical and health sciences ,Mice ,030104 developmental biology ,0302 clinical medicine ,DNA methylation ,Illumina Methylation Assay ,Animals ,Sulfites ,CpG Islands ,Methylated DNA immunoprecipitation ,Single-Cell Analysis ,030217 neurology & neurosurgery ,Epigenomics - Abstract
DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.
- Published
- 2017
28. Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine
- Author
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Cynthia J. Burrows, Aaron M. Fleming, and Yun Ding
- Subjects
Electrophoresis, Agar Gel ,0301 basic medicine ,Sanger sequencing ,DNA nanoball sequencing ,Guanine ,Base pair ,Sequence Analysis, DNA ,Formamidopyrimidine DNA glycosylase ,Base excision repair ,Biology ,010402 general chemistry ,Polymerase Chain Reaction ,01 natural sciences ,Molecular biology ,Article ,DNA sequencing ,0104 chemical sciences ,Sequencing by ligation ,03 medical and health sciences ,genomic DNA ,symbols.namesake ,030104 developmental biology ,symbols ,Base Pairing ,Oxidation-Reduction - Abstract
The DNA base guanine (G) can be oxidatively modified to 8-oxo-7,8-dihydroguanine (OG). Extraction of genomic DNA followed by nuclease digestion and mass spectrometry analysis have found OG is present at background levels of ~1 out of 106 Gs; however, this approach cannot determine the locations for the OGs in the genome. Thus, in this methods report, we outline three different methods (A, B, and C) for sequencing OG in DNA. Method A sequences OG by utilizing the base excision repair pathway to delete the OG nucleotide from the DNA that is then detected by Sanger sequencing as a deletion signature. Method B sequences OG by harnessing the base excision repair pathway to convert OG to an unnatural DNA base pair followed by Sanger sequencing to locate the unnatural base pair indicating where OG was located. Method C (i.e., OG-Seq) takes genomic DNA sheared to ~150 bps followed by selectively biotinylating the OG-containing fragments for affinity purification and enrichment of the OG-modified strands. The OG-modified fragments are sequenced on a next-generation sequencing platform to locate OG on the genomic scale with a resolution of ~150 bps. The methods outlined are then compared and contrasted allowing researchers to select the one that best suits their experimental goals.
- Published
- 2017
29. Methods for Assessing DNA Cytosine Modifications Genome-Wide
- Author
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Gerd P. Pfeifer and Tibor A. Rauch
- Subjects
Genetics ,DNA nanoball sequencing ,5-Methylcytosine ,chemistry.chemical_compound ,Sequencing by hybridization ,chemistry ,Bisulfite sequencing ,Methylated DNA immunoprecipitation ,Biology ,DNA sequencing ,Sequencing by ligation ,Epigenomics - Abstract
5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most frequent cytosine modifications in mammalian DNA. Various approaches have been developed and employed to analyze DNA cytosine modification patterns in genomic DNA. These techniques are based on common principles, including methylation-sensitive restriction enzymes, sodium bisulfite conversion of genomic DNA, and affinity capture with either antibodies or proteins that bind specifically to methylated CpG sequences. For many applications, high-throughput sequencing approaches have replaced microarray platforms. These methods are useful to analyze the entire genome at various levels of resolution or can be designed to focus on the most critical genomic loci. In this chapter, we summarize and discuss several of these technologies in detail.
- Published
- 2017
30. Cat-D: a targeted sequencing method for the simultaneous detection of small DNA mutations and large DNA deletions with flexible boundaries
- Author
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Li-Feng Zhang, Udita Chandola, Ru Hong, and School of Biological Sciences
- Subjects
0301 basic medicine ,DNA nanoball sequencing ,Genotype ,Population ,lcsh:Medicine ,Biology ,medicine.disease_cause ,Article ,DNA sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,alpha-Thalassemia ,Sequencing by hybridization ,medicine ,Humans ,Point Mutation ,lcsh:Science ,education ,Sequence Deletion ,Genetics ,DNA Mutations ,education.field_of_study ,Mutation ,Multidisciplinary ,lcsh:R ,DNA ,Sequence Analysis, DNA ,genomic DNA ,030104 developmental biology ,chemistry ,lcsh:Q ,Targeted DNA Sequencing Method ,030217 neurology & neurosurgery - Abstract
We developed a targeted DNA sequencing method that is capable of detecting a comprehensive panel of DNA mutations including small DNA mutations and large DNA deletions with unknown/flexible boundaries. The method directly identifies the large DNA deletions (Cat-D) without relying on sequencing coverage to make the genotype calls. We performed the method to simultaneously detect 10 small DNA mutations in β-thalassemia and 2 large genomic deletions in α-thalassemia from 10 genomic DNA samples. Cat-D was performed on 8 genomic DNA samples in duplicate. The 18 Cat-D samples were combined in one sequencing run. In total, 216 genotype calls were made, and 215 of the genotype calls were accurate. No false negative genotype calls were made. One false positive genotype call was made on one target mutation in one experimental duplicate from a genomic DNA sample. In summary, Cat-D can be developed into a robust, high-throughput and cost-effective method suitable for population-based carrier screens. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version
- Published
- 2017
31. Transgene Structure Analysis of Genetically Modified OsCK1 Rice : Whole Genome Shotgun Sequencing as a Tool for Structure Analysis of Multi-Inserted T-DNAs
- Author
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Yang Qin, Jin-Hyung Lee, Kong-Sik Shin, Soon-Jong Kweon, He-Jong Woo, Myung Ho Lim, and Hong-Il Ahn
- Subjects
Cancer genome sequencing ,Genetics ,Sequence-tagged site ,DNA nanoball sequencing ,Shotgun sequencing ,Genomics ,Genomic library ,Genome project ,Biology ,DNA sequencing - Published
- 2014
32. Reading Single DNA with DNA Polymerase Followed by Atomic Force Microscopy
- Author
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Jeong Soo Lee, Joung Hun Kim, Youngkyu Kim, Yoonhee Lee, Eung-Sam Kim, Joon Won Park, Bong Chu Shim, and Seong Moon Cho
- Subjects
DNA nanoball sequencing ,Base Sequence ,biology ,DNA polymerase ,Chemistry ,DNA ,DNA-Directed DNA Polymerase ,General Chemistry ,Microscopy, Atomic Force ,Polymerase cycling assembly ,Biochemistry ,Molecular biology ,Catalysis ,DNA sequencing ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,biology.protein ,Biophysics ,A-DNA ,Polymerase ,Single molecule real time sequencing - Abstract
The importance of DNA sequencing in the life sciences and personalized medicine is continually increasing. Single-molecule sequencing methods have been developed to analyze DNA directly without the need for amplification. Here, we present a new approach to sequencing single DNA molecules using atomic force microscopy (AFM). In our approach, four surface-conjugated nucleotides were examined sequentially with a DNA polymerase-immobilized AFM tip. By observing the specific rupture events upon examination of a matching nucleotide, we could determine the template base bound in the polymerase's active site. The subsequent incorporation of the complementary base in solution enabled the next base to be read. Additionally, we observed that the DNA polymerase could incorporate the surface-conjugated dGTP when the applied force was controlled by employing the force-clamp mode.
- Published
- 2014
33. Decoding long nanopore sequencing reads of natural DNA
- Author
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Riza M. Daza, Andrew Adey, Kenji Doering, Jonathan M. Craig, Jenny Mae Samson, Kyle W. Langford, Ian M. Derrington, Henry Brinkerhoff, Jay Shendure, Andrew H. Laszlo, Jens H. Gundlach, Ian C. Nova, and Brian C. Ross
- Subjects
DNA nanoball sequencing ,Biomedical Engineering ,Phi X 174 ,Sequence assembly ,Bioengineering ,Hybrid genome assembly ,02 engineering and technology ,Computational biology ,Applied Microbiology and Biotechnology ,Genome ,Article ,Nanopores ,03 medical and health sciences ,030304 developmental biology ,Genetics ,0303 health sciences ,biology ,DNA ,Sequence Analysis, DNA ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Nanopore ,Molecular Medicine ,Nanopore sequencing ,0210 nano-technology ,Biotechnology ,Reference genome - Abstract
Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length that can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides the foundation for nanopore sequencing of long, complex, natural DNA strands.
- Published
- 2014
34. Capture and Amplification by Tailing and Switching (CATS)
- Author
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Peter Lichter, Marc Zapatka, Andrius Serva, Barbara Burwinkel, Andrey Turchinovich, and Harald Surowy
- Subjects
DNA nanoball sequencing ,Time Factors ,Massive parallel sequencing ,Sequence Analysis, RNA ,Technical Paper ,Oligonucleotide ,Multiple displacement amplification ,High-Throughput Nucleotide Sequencing ,DNA ,Sequence Analysis, DNA ,Cell Biology ,Computational biology ,Biology ,Molecular biology ,DNA sequencing ,Sequencing by ligation ,Sequencing by hybridization ,Cell Line, Tumor ,Animals ,Humans ,RNA ,Genomic library ,Caenorhabditis elegans ,Molecular Biology ,Gene Library - Abstract
Massive parallel sequencing (MPS) technologies have paved the way into new areas of research including individualized medicine. However, sequencing of trace amounts of DNA or RNA still remains a major challenge, especially for degraded nucleic acids like circulating DNA. This together with high cost and time requirements impedes many important applications of MPS in medicine and fundamental science. We have established a fast, cheap and highly efficient protocol called ‘Capture and Amplification by Tailing and Switching’ (CATS) to directly generate ready-to-sequence libraries for MPS from nanogram and picogram quantities of both DNA and RNA. Furthermore, those DNA libraries are strand-specific, can be prepared within 2–3 h and do not require preliminary sample amplification steps. To exemplify the capacity of the technique, we have generated and sequenced DNA libraries from hundred-picogram amounts of circulating nucleic acids isolated from human blood plasma, one nanogram of mRNA-enriched total RNA from cultured cells and few nanograms of bisulfite-converted DNA. The approach for DNA library preparation from minimal and fragmented input described here will find broad application in diverse research areas such as translational medicine including therapy monitoring, prediction, prognosis and early detection of various human disorders and will permit high-throughput DNA sequencing from previously inaccessible material such as minute forensic and archeological samples.
- Published
- 2014
35. Microarray-based resonance light scattering assay for detecting DNA methylation and human DNA methyltransferase simultaneously with high sensitivity
- Author
-
Zhenxin Wang, Lan Ma, Min Su, and Tao Li
- Subjects
DNA (Cytosine-5-)-Methyltransferase 1 ,DNA nanoball sequencing ,Methyltransferase ,Light ,Metal Nanoparticles ,Biochemistry ,Analytical Chemistry ,chemistry.chemical_compound ,Limit of Detection ,Electrochemistry ,Humans ,Scattering, Radiation ,Environmental Chemistry ,DNA (Cytosine-5-)-Methyltransferases ,Methylated DNA immunoprecipitation ,Spectroscopy ,Enzyme Assays ,Chemistry ,DNA ,Equipment Design ,DNA Methylation ,Microarray Analysis ,Molecular biology ,Restriction enzyme ,DNA methylation ,DNMT1 ,Illumina Methylation Assay ,Gold - Abstract
A microarray-based resonance light scattering assay, with the combination of methylation-sensitive endonuclease and gold nanoparticle (GNP) probes, has been proposed to sensitively distinguish the DNA methylation level as low as 0.01% (10 pM methylated DNA in 100 nM total DNA) and detect human DNA methyltransferase 1 (Dnmt1) down to 0.1 U mL(-1).
- Published
- 2014
36. MethSMRT: an integrative database for DNA N6-methyladenine and N4-methylcytosine generated by single-molecular real-time sequencing
- Author
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Pohao Ye, Yizhi Liu, Kai-Ning Chen, Zhi Xie, Chuan-Le Xiao, and Yizhao Luan
- Subjects
0301 basic medicine ,DNA nanoball sequencing ,Genome browser ,Biology ,computer.software_genre ,Polymorphism, Single Nucleotide ,03 medical and health sciences ,Cytosine ,Genetics ,Animals ,Database Issue ,RNA-Directed DNA Methylation ,Epigenomics ,Genome ,Database ,Adenine ,DNA ,Sequence Analysis, DNA ,DNA Methylation ,Sequencing by ligation ,030104 developmental biology ,DNA methylation ,Illumina Methylation Assay ,Databases, Nucleic Acid ,computer ,Single molecule real time sequencing - Abstract
DNA methylation is an important type of epigenetic modifications, where 5- methylcytosine (5mC), 6-methyadenine (6mA) and 4-methylcytosine (4mC) are the most common types. Previous efforts have been largely focused on 5mC, providing invaluable insights into epigenetic regulation through DNA methylation. Recently developed single-molecule real-time (SMRT) sequencing technology provides a unique opportunity to detect the less studied DNA 6mA and 4mC modifications at single-nucleotide resolution. With a rapidly increased amount of SMRT sequencing data generated, there is an emerging demand to systematically explore DNA 6mA and 4mC modifications from these data sets. MethSMRT is the first resource hosting DNA 6mA and 4mC methylomes. All the data sets were processed using the same analysis pipeline with the same quality control. The current version of the database provides a platform to store, browse, search and download epigenome-wide methylation profiles of 156 species, including seven eukaryotes such as Arabidopsis, C. elegans, Drosophila, mouse and yeast, as well as 149 prokaryotes. It also offers a genome browser to visualize the methylation sites and related information such as single nucleotide polymorphisms (SNP) and genomic annotation. Furthermore, the database provides a quick summary of statistics of methylome of 6mA and 4mC and predicted methylation motifs for each species. MethSMRT is publicly available at http://sysbio.sysu.edu.cn/methsmrt/ without use restriction.
- Published
- 2016
37. A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants
- Author
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Rocío B. Rodríguez-Estrada, John M. Williams, Aracely García-Bravo, Thuluz Meza-Menchaca, Aracely López-Monteon, Rossana C. Zepeda, and Angel Ramos-Ligonio
- Subjects
DNA nanoball sequencing ,Microarray ,DNA Mutational Analysis ,Biology ,lcsh:Chemical technology ,HPV-18 ,Polymorphism, Single Nucleotide ,Sensitivity and Specificity ,Biochemistry ,Article ,Analytical Chemistry ,chemistry.chemical_compound ,lcsh:TP1-1185 ,Electrical and Electronic Engineering ,Instrumentation ,Oligonucleotide Array Sequence Analysis ,Genetics ,variants ,Human papillomavirus 18 ,Phylogenetic tree ,Oligonucleotide ,Point mutation ,Hybridization probe ,Genetic Variation ,Reproducibility of Results ,Equipment Design ,Atomic and Molecular Physics, and Optics ,Equipment Failure Analysis ,chemistry ,LCR ,DNA, Viral ,DNA Probes ,microarray ,DNA - Abstract
We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18’s variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings.
- Published
- 2013
38. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
- Author
-
Jennifer A. Doudna, Vikram Pattanayak, Steven Lin, David R. Liu, John Paul Guilinger, and Enbo Ma
- Subjects
DNA nanoball sequencing ,Streptococcus pyogenes ,Biomedical Engineering ,Bioengineering ,Biology ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,chemistry.chemical_compound ,Endonuclease ,0302 clinical medicine ,Bacterial Proteins ,Humans ,Guide RNA ,030304 developmental biology ,0303 health sciences ,Nuclease ,Genome ,Cas9 ,High-Throughput Nucleotide Sequencing ,RNA ,DNA ,Genomics ,Sequence Analysis, DNA ,Endonucleases ,Molecular biology ,HEK293 Cells ,chemistry ,biology.protein ,Molecular Medicine ,Human genome ,Genetic Engineering ,030217 neurology & neurosurgery ,RNA, Guide, Kinetoplastida ,Biotechnology - Abstract
The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.
- Published
- 2013
39. Fluorescence Imaging of Single-Copy DNA Sequences within the Human Genome Using PNA-Directed Padlock Probe Assembly
- Author
-
Anastasia I. Yaroslavsky and Irina V. Smolina
- Subjects
Peptide Nucleic Acids ,DNA nanoball sequencing ,Clinical Biochemistry ,Biology ,010402 general chemistry ,DNA, Mitochondrial ,01 natural sciences ,Biochemistry ,Article ,DNA sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,Nucleic acid thermodynamics ,Drug Discovery ,Humans ,Molecular Biology ,030304 developmental biology ,Pharmacology ,0303 health sciences ,Base Sequence ,Peptide nucleic acid ,Genome, Human ,Hybridization probe ,Optical Imaging ,Nucleic Acid Hybridization ,DNA ,General Medicine ,Molecular biology ,0104 chemical sciences ,genomic DNA ,chemistry ,Rolling circle replication ,Genome, Mitochondrial ,Molecular Medicine ,Human genome ,Oligonucleotide Probes - Abstract
SummaryWe present an approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single-copy sensitivity and single-base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this technique by successfully detecting six single-copy target sites on human mitochondrial and autosomal DNA. We also demonstrate the high selectivity of this method by detecting X- and Y-specific sequences on human sex chromosomes and by simultaneously detecting three sequence-specific target sites. Finally, we discriminate two target sites that differ by 2 nt. The PNA-RCA-FISH approach is a distinctive in situ hybridization method capable of multitarget visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific.
- Published
- 2013
40. Direct Detection of Bacterial Genomic DNA at Sub-Femtomolar Concentrations Using Single Molecule Arrays
- Author
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Dandan Shan, Linan Song, Ryan B. Hayman, Sean Sullivan, Brian A. Pink, Melissa Gardel, Lyndsey York, David C. Duffy, Kaitlin A. Minnehan, Mingwei Zhao, Aaron F. Phillips, and David R. Walt
- Subjects
DNA, Bacterial ,Detection limit ,DNA nanoball sequencing ,biology ,Chemistry ,Molecular biology ,Analytical Chemistry ,law.invention ,genomic DNA ,Restriction enzyme ,chemistry.chemical_compound ,law ,biology.protein ,Humans ,Nanotechnology ,Genome, Bacterial ,Polymerase ,Polymerase chain reaction ,DNA ,Oligonucleotide Array Sequence Analysis ,Single molecule real time sequencing - Abstract
We report a method for the sensitive measurement of genomic DNA based on the direct detection of single molecules of DNA in arrays of femtoliter wells. The method begins by generating short fragments of DNA from large, double-stranded molecules of genomic DNA using either restriction enzymes or sonication. Single-stranded fragments are then generated by melting the duplex, and these fragments are hybridized to complementary biotinylated detection probes and capture probes on paramagnetic beads. The resulting DNA complexes are then labeled with an enzyme (streptavidin-β-galactosidase), and single enzymes associated with these complexes on beads are detected in single molecule arrays (Simoa). DNA concentration is quantified by determining the average number of enzymes per bead via Poisson statistics (digital) or the average bead intensity (analog). The Simoa DNA assay was used to detect genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, or 2100 DNA molecules per 50 μL sample. We used this assay to detect S. aureus spiked into (a) whole blood, with an average LOD of 1100 bacteria per 25 μL sample (0.074 fM), and (b) water from the Charles River, with an LOD of 1300 bacteria per 50 μL sample (0.042 fM). Bacteria were detected in river water without prior purification of DNA. The Simoa DNA assay, which directly detects target DNA molecules without molecular replication, is an attractive alternative to existing sensitive DNA detection technologies that rely on amplification using polymerases, such as the polymerase chain reaction (PCR).
- Published
- 2013
41. Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing
- Author
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Omar Jabado, Richard J. Roberts, Gintaras Deikus, Michael C. Chao, Steve W. Turner, Anjali Mandlik, Bojan Losic, Diana Munera, Brigid M. Davis, Milind Mahajan, Vipin Kumar, Khai Luong, Gang Fang, Zhixing Feng, Jonas Korlach, Tyson A. Clark, Andrew Chess, Onureena Banerjee, David I. Friedman, Iain A. Murray, Matthew K. Waldor, Alona Keren-Paz, and Eric E. Schadt
- Subjects
Genetics ,DNA nanoball sequencing ,Biomedical Engineering ,Bioengineering ,Biology ,Applied Microbiology and Biotechnology ,DNA Restriction-Modification Enzymes ,Genome ,DNA sequencing ,Sequencing by hybridization ,DNA methylation ,Molecular Medicine ,Biotechnology ,Single molecule real time sequencing ,Epigenomics - Abstract
Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.
- Published
- 2012
42. A New Trace Analytic Method for the Determination of Sequence-Specific DNA with Fluorescent Probes
- Author
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Guo-Ping Zeng, Dong-Shan Xiang, and Jin-Zhang Cai
- Subjects
DNA nanoball sequencing ,Oligonucleotide ,Biochemistry (medical) ,Clinical Biochemistry ,Fluorescence spectrometry ,Biochemistry ,Molecular biology ,Fluorescence ,Analytical Chemistry ,chemistry.chemical_compound ,Nucleic acid thermodynamics ,chemistry ,Relative fluorescence units ,Electrochemistry ,Biophysics ,Spectroscopy ,DNA ,Single molecule real time sequencing - Abstract
A new method of fluorescence spectrometry detection of single-strand DNA (ssDNA) was established by hybridizing the ssDNA with its complementary ssDNA to form double-stranded DNA (dsDNA). Our results show that the fluorescence intensity increased significantly when the nucleic acid molecular “light switch"(Ru(phen)2dppx2+) or Hoechst 33258 dye interacted with dsDNA, and the fluorescence intensity also increased as the DNA concentration increased. The changing law was also studied about how the fluorescence intensity changed when the two kinds of fluorescent probes interacted with oligonucleotide of different lengths and different sequences, as well as DNA-DNA′ hybridization products. Then, the effect of the bases mismatch, varying length of DNA chain, and different DNA sequences on the fluorescence intensity were explored at the same time, by detecting the specific DNA sequence of avian influenza H1N1 virus, cauliflower mosaic virus, and hepatitis C virus. Additionally, the selectivity, linear range, and ...
- Published
- 2012
43. Fluorescence detection of single-nucleotide differences using aptamer-forming binary DNA probes
- Author
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Saori Kamoto, Masumi Hyuga, and Teru Kato
- Subjects
DNA nanoball sequencing ,Genotyping Techniques ,Aptamer ,Cholic Acid ,010402 general chemistry ,Molecular Inversion Probe ,01 natural sciences ,Biochemistry ,Polymorphism, Single Nucleotide ,Analytical Chemistry ,chemistry.chemical_compound ,Nucleic acid thermodynamics ,Electrochemistry ,Environmental Chemistry ,Humans ,Spectroscopy ,010405 organic chemistry ,Hybridization probe ,Nucleic Acid Hybridization ,DNA ,Aptamers, Nucleotide ,Molecular biology ,0104 chemical sciences ,SNP genotyping ,Cytochrome P-450 CYP2C19 ,chemistry ,Biophysics ,DNA Probes - Abstract
We report a simple method for fluorescence detection of single-nucleotide alterations in a long target DNA, which is based on the formation of a three-way-junction-structured cholic-acid-binding DNA aptamer by the hybridization of the target with binary DNA probes. The new method was successfully exploited for SNP genotyping of human CYP2C19 gene.
- Published
- 2016
44. Single molecule targeted sequencing for cancer gene mutation detection
- Author
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Liu Song, Deng Liwei, Yongqian Gao, Li Gailing, Wu Zengding, Michael W. Deem, Jiankui He, Wu Ping, Jinsen Cai, Ji Daorui, Zhao Luyang, Ge Liangjin, Gao Yan, Yan Qin, and Huan Jin
- Subjects
0301 basic medicine ,Cancer genome sequencing ,Genetics ,DNA nanoball sequencing ,Multidisciplinary ,Massive parallel sequencing ,High-Throughput Nucleotide Sequencing ,Sequence Analysis, DNA ,Biology ,DNA sequencing ,Article ,Massively parallel signature sequencing ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Single cell sequencing ,030220 oncology & carcinogenesis ,Neoplasms ,Mutation ,Humans ,Pathology, Molecular ,Exome sequencing ,Illumina dye sequencing ,Genes, Neoplasm - Abstract
With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis.
- Published
- 2016
45. Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules
- Author
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Baptiste Mayjonade, Jérôme Gouzy, Cécile Donnadieu, Caroline Callot, Nicolas B. Langlade, Stéphane Muños, William Marande, Nicolas Pouilly, Centre National de la Recherche Scientifique (CNRS), Laboratoire des interactions plantes micro-organismes (LIPM), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Génétique Physiologie et Systèmes d'Elevage (GenPhySE ), École nationale supérieure agronomique de Toulouse [ENSAT]-Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Centre National de Ressources Génomiques Végétales (CNRGV), Institut National de la Recherche Agronomique (INRA), Centre de Recherches en Cancérologie de Toulouse (CRCT), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM), Université Fédérale Toulouse Midi-Pyrénées, SUNRISE project [ANR-11-BTBR-0005], Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des Interactions Plantes Micro organismes, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-École nationale supérieure agronomique de Toulouse [ENSAT]
- Subjects
0106 biological sciences ,DNA, Bacterial ,0301 basic medicine ,DNA nanoball sequencing ,DNA, Plant ,[SDV]Life Sciences [q-bio] ,Sequence assembly ,Computational biology ,Biology ,01 natural sciences ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Sequencing by hybridization ,Animals ,Molecule ,Protocol (object-oriented programming) ,DNA extraction ,PacBio ,Massive parallel sequencing ,Genome ,Shotgun sequencing ,Chemistry ,Extraction (chemistry) ,High-Throughput Nucleotide Sequencing ,Ion semiconductor sequencing ,DNA ,Sequence Analysis, DNA ,Sequencing by ligation ,Molecular Weight ,DNA sequencer ,genomic DNA ,030104 developmental biology ,long-read sequencing ,010606 plant biology & botany ,Biotechnology - Abstract
De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb.
- Published
- 2016
46. Eukaryotic Single-Cell mRNA Sequencing
- Author
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Keith E. Szulwach and Kenneth J. Livak
- Subjects
DNA nanoball sequencing ,genomic DNA ,MRNA Sequencing ,Massive parallel sequencing ,Genomics ,Computational biology ,Biology ,DNA sequencing ,Illumina dye sequencing ,Sequencing by ligation - Abstract
Single-cell DNA sequencing is a rapidly developing field that holds great potential for revolutionizing our understanding of how cellular heterogeneity contributes to the initiation and progression of disease. Methods in single-cell genomics have already emerged as transformative techniques enabling the characterization of heterogeneous cell populations in cancer, neurobiology, immunology, and normal development with unprecedented detail. The ability to isolate individual cells followed by amplification of genomic DNA and high-throughput sequencing enables the identification of co-occurring somatic mutations, an outcome not obtainable by conventional analysis of DNA isolated from many cells. As a result, phylogenetic relationships can be accurately discerned in order to reveal the evolutionary history of cell populations as new mutations are acquired and distinct phenotypes arise. In this chapter, we describe current methods in single-cell DNA sequencing. We summarize the typical approaches to genome-scale sequencing of amplified DNA and the analytical methods for processing such datasets. Statistical approaches for distinguishing mutations from technical artifacts generated during amplification of genomic DNA, determining the clonality of heterogeneous cell populations, and discerning phylogenetic relationships between clones are also described.
- Published
- 2016
47. Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules
- Author
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Olivier Hyrien, Vincent Gaggioli, Francesco De Carli, Gaël A. Millot, Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge [UK] (CAM), Dynamique de l'information génétique : bases fondamentales et cancer (DIG CANCER), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Sorbonne Université (SU), This work was supported by the Ligue Nationale contre le Cancer (Comité de Paris), the Fondation ARC pour la recherche sur le cancer and the Agence Nationale pour la Recherche (ANR-15-CE12-0011-01). FDC was supported by a Ph-D fellowship from the Université Pierre et Marie Curie., ANR-15-CE12-0011,LIGHTCOMB,Méthodes de peignage moléculaire à haut débit pour une cartographie rapide de la réplication du génome humain(2015), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), millot, gael, Méthodes de peignage moléculaire à haut débit pour une cartographie rapide de la réplication du génome humain - - LIGHTCOMB2015 - ANR-15-CE12-0011 - AAPG2015 - VALID, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), and Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,DNA Replication ,Embryology ,DNA nanoball sequencing ,[SDV]Life Sciences [q-bio] ,MESH: DNA Replication ,In situ hybridization ,Fluorescence ,03 medical and health sciences ,chemistry.chemical_compound ,Endonuclease ,Xenopus laevis ,MESH: Xenopus laevis ,MESH: Endonucleases ,MESH: Cell-Free System ,Fluorescence microscope ,Animals ,DNA Barcoding, Taxonomic ,Nucleotide ,MESH: Animals ,MESH: DNA Barcoding, Taxonomic ,chemistry.chemical_classification ,biology ,Cell-Free System ,Hybridization probe ,MESH: Fluorescence ,DNA replication ,Endonucleases ,Molecular biology ,[SDV] Life Sciences [q-bio] ,030104 developmental biology ,chemistry ,biology.protein ,DNA ,Developmental Biology - Abstract
International audience; DNA combing is a standard technique to map DNA replication at the single molecule level. Typically, replicating DNA is metabolically labelled with nucleoside or nucleotide analogs, purified, stretched on coverslips and treated with fluorescent antibodies to reveal tracts of newly synthesized DNA. Fibres containing a locus of interest can then be identified by fluorescent in situ hybridization (FISH) with DNA probes. These steps are complex and the throughput is low. Here, we describe a simpler, antibody-free method to reveal replication tracts and identify the locus of origin of combed DNA replication intermediates. DNA was replicated in Xenopus egg extracts in the presence of a fluorescent dUTP. Purified DNA was barcoded by nicking with Nt.BspQI, a site-specific nicking endonuclease (NE), followed by limited nick-translation in the presence of another fluorescent dUTP. DNA was then stained with YOYO-1, a fluorescent DNA intercalator, and combed. Direct epifluorescence revealed the DNA molecules, their replication tracts and their Nt.BspQI sites in three distinct colours. Replication intermediates could thus be aligned to a reference genome map. In addition, replicated DNA segments showed a stronger YOYO-1 fluorescence than unreplicated segments. The entire length, replication tracts, and NE sites of combed DNA molecules can be simultaneously visualized in three distinct colours by standard epifluorescence microscopy, with no need for antibody staining and/or FISH detection. Furthermore, replication bubbles can be detected by quantitative YOYO-1 staining, eliminating the need for metabolic labelling. These results provide a starting point for genome-wide, single-molecule mapping of DNA replication in any organism.
- Published
- 2016
48. Single Labeled DNA FIT Probes for Avoiding False-Positive Signaling in the Detection of DNA/RNA in qPCR or Cell Media
- Author
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Felix Hövelmann, Lucas Bethge, and Oliver Seitz
- Subjects
DNA nanoball sequencing ,Base pair ,Biology ,Real-Time Polymerase Chain Reaction ,Biochemistry ,Madin Darby Canine Kidney Cells ,Nucleic acid thermodynamics ,Dogs ,Sequencing by hybridization ,Molecular beacon ,Animals ,Benzothiazoles ,Molecular Biology ,Polymerase ,Base Sequence ,Oligonucleotide ,Hybridization probe ,Organic Chemistry ,Nucleic Acid Hybridization ,DNA ,Intercalating Agents ,Spectrometry, Fluorescence ,Quinolines ,biology.protein ,RNA ,Molecular Medicine ,Oligonucleotide Probes - Abstract
Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real-time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance-dependent interaction between a fluorophore and another label. Such dual-labeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label-label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, "DNA FIT probes", that are designed to avoid false-positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the "TO base" in cis with the disordered nucleobases of the single strand, and 2) intercalation of the "TO nucleotide" with double strands in trans. However, formation of the probe-target duplex enforces stacking and increases the fluorescence of the TO base. We explored open-chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe-target complexes. DNA and RNA targets provided up to 12-fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false-positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.
- Published
- 2012
49. Metrological support for DNA sequencing
- Author
-
A. V. Mardanov, N. V. Ravin, S. A. Kononogov, Yu. A. Kudeyarov, S. S. Golubev, P. Yu. Nikolaeva, and Konstantin G. Skryabin
- Subjects
DNA nanoball sequencing ,Computer science ,Shotgun sequencing ,Applied Mathematics ,Nucleic acid sequence ,Pyrosequencing ,Ion semiconductor sequencing ,Computational biology ,Instrumentation ,DNA sequencing ,Illumina dye sequencing ,Sequencing by ligation - Abstract
We discuss the operating principle of a pyrosequencer, which is an instrument for sequential reading of the nucleotide sequence of DNA molecules in pyrophosphate sequencing (pyrosequencing). We formulate the basic principles of metrological support for DNA sequencing and we present the results of studies of the metrological characteristics of the GS FLX pyrosequencer. We list the major problems encountered in designing a system for metrological support of DNA sequencing.
- Published
- 2012
50. Designing of sequencing assay assisted by capillary electrophoresis based on DNA folding analysis: An application to the VCAM1 gene
- Author
-
Marco Orrù, Bastiana Arcadu, R Piga, and Germano Orrù
- Subjects
DNA nanoball sequencing ,Clinical Biochemistry ,Vascular Cell Adhesion Molecule-1 ,Computational biology ,Biology ,Real-Time Polymerase Chain Reaction ,Polymorphism, Single Nucleotide ,Biochemistry ,Deep sequencing ,Analytical Chemistry ,symbols.namesake ,Sequencing by hybridization ,Primer walking ,Humans ,Genetics ,Sanger sequencing ,Shotgun sequencing ,Computational Biology ,Electrophoresis, Capillary ,DNA ,Exons ,Sequence Analysis, DNA ,Sequencing by ligation ,DNA sequencer ,symbols ,Nucleic Acid Conformation ,Thermodynamics - Abstract
In this work, we describe a fast standardized molecular method for DNA sequencing assisted by capillary electrophoresis with a particular emphasis on bioinformatic approaches to avoid sequencing errors due to complex DNA regions. In this case, the method was applied on the human vascular adhesion molecule 1 (VCAM1) gene. VCAM1 sequence, in fact, shows many thermodynamically critical parameters such as very low GC content (30-40%), many nucleotide stack areas, i.e. hairpins, self-complementary regions. With a traditional primer design approach it was difficult to design correct PCR oligonucleotides, thus sometimes, the chromatogram showed an illegible profile. By a strategy involving various bioinformatic tools (Mfold, Oligo, Highter), we investigated the role of the DNA-folding analysis in the assistance of primer design for the DNA sequencing of fragments with high -ΔG stem-loop regions. This new approach allowed us to sequence nine different VCAM1 regions each containing the respective exon. Our results, based on different DNA samples recruited from oral brushes taken from ten different subjects, identified four different SNPs (c.662-7C/T, c.1793-79A>G, c.2079C/T, c.2208A>G) with high reproducibility.
- Published
- 2012
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