scholarly journals Enzyme-free optical DNA mapping of the human genome using competitive binding

2019 ◽  
Vol 47 (15) ◽  
pp. e89-e89 ◽  
Author(s):  
Vilhelm Müller ◽  
Albertas Dvirnas ◽  
John Andersson ◽  
Vandana Singh ◽  
Sriram KK ◽  
...  
Keyword(s):  
Dna Damage ◽  
Human Genome ◽  
Long Range ◽  
White Blood Cells ◽  
Competitive Binding ◽  
Sequence Information ◽  
Dna Molecules ◽  
Structural Variations ◽  
Artificial Chromosomes ◽  
Dna Mapping

Abstract Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized.

scholarly journals Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Lena K. Nyberg ◽  
Saair Quaderi ◽  
Gustav Emilsson ◽  
Nahid Karami ◽  
Erik Lagerstedt ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Bacterial Resistance ◽  
Clinical Epidemiology ◽  
Rapid Identification ◽  
Sequence Information ◽  
Dna Molecules ◽  
Structural Variations ◽  
Mapping Procedure ◽  
Single Dna Molecules ◽  
Nanofluidic Channels

Abstract The rapid spread of antibiotic resistance – currently one of the greatest threats to human health according to WHO – is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.

scholarly journals Combining dense and sparse labeling in optical DNA mapping

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260489
Author(s):  
Erik Torstensson ◽  
Gaurav Goyal ◽  
Anna Johnning ◽  
Fredrik Westerlund ◽  
Tobias Ambjörnsson
Keyword(s):  
Structural Changes ◽  
Competitive Binding ◽  
Fluorescent Labeling ◽  
P Value ◽  
Dna Barcodes ◽  
Dna Molecules ◽  
P Values ◽  
Dna Labeling ◽  
Dna Mapping ◽  
Labeling Schemes

Optical DNA mapping (ODM) is based on fluorescent labeling, stretching and imaging of single DNA molecules to obtain sequence-specific fluorescence profiles, DNA barcodes. These barcodes can be mapped to theoretical counterparts obtained from DNA reference sequences, which in turn allow for DNA identification in complex samples and for detecting structural changes in individual DNA molecules. There are several types of DNA labeling schemes for ODM and for each labeling type one or several types of match scoring methods are used. By combining the information from multiple labeling schemes one can potentially improve mapping confidence; however, combining match scores from different labeling assays has not been implemented yet. In this study, we introduce two theoretical methods for dealing with analysis of DNA molecules with multiple label types. In our first method, we convert the alignment scores, given as output from the different assays, into p-values using carefully crafted null models. We then combine the p-values for different label types using standard methods to obtain a combined match score and an associated combined p-value. In the second method, we use a block bootstrap approach to check for the uniqueness of a match to a database for all barcodes matching with a combined p-value below a predefined threshold. For obtaining experimental dual-labeled DNA barcodes, we introduce a novel assay where we cut plasmid DNA molecules from bacteria with restriction enzymes and the cut sites serve as sequence-specific markers, which together with barcodes obtained using the established competitive binding labeling method, form a dual-labeled barcode. All experimental data in this study originates from this assay, but we point out that our theoretical framework can be used to combine data from all kinds of available optical DNA mapping assays. We test our multiple labeling frameworks on barcodes from two different plasmids and synthetically generated barcodes (combined competitive-binding- and nick-labeling). It is demonstrated that by simultaneously using the information from all label types, we can substantially increase the significance when we match experimental barcodes to a database consisting of theoretical barcodes for all sequenced plasmids.

Thermoplastic Nanofluidic Devices for Identifying Abasic Sites in Single DNA Molecules

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Steven A Soper ◽  
Swarnagowri Vaidyanathan ◽  
Franklin Uba ◽  
Bo Hu ◽  
David Kaufman ◽  
...  
Keyword(s):  
Dna Damage ◽  
Therapeutic Efficacy ◽  
Double Strand Breaks ◽  
Dna Molecules ◽  
Strand Breaks ◽  
Single Dna Molecules ◽  
Abasic Sites ◽  
Nanofluidic Devices ◽  
Ap Sites

DNA damage can take many forms such as double-strand breaks and/or the formation of abasic (apurinic/apyrimidinic; AP) sites. The presence of AP sites can be used to determine therapeutic efficacy...

scholarly journals Identification and Characterization of Novel Human Endogenous Retrovirus Families by Phylogenetic Screening of the Human Genome Mapping Project Database

2000 ◽  
Vol 74 (8) ◽  
pp. 3715-3730 ◽  
Author(s):  
Michael Tristem
Keyword(s):  
Human Genome ◽  
Genome Mapping ◽  
Sequence Data ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Human Endogenous Retrovirus ◽  
Sequence Information ◽  
Class Iii ◽  
Genome Mapping Project ◽  
Human Genome Mapping Project

ABSTRACT Human endogenous retroviruses (HERVs) were first identified almost 20 years ago, and since then numerous families have been described. It has, however, been difficult to obtain a good estimate of both the total number of independently derived families and their relationship to each other as well as to other members of the familyRetroviridae. In this study, I used sequence data derived from over 150 novel HERVs, obtained from the Human Genome Mapping Project database, and a variety of recently identified nonhuman retroviruses to classify the HERVs into 22 independently acquired families. Of these, 17 families were loosely assigned to the class I HERVs, 3 to the class II HERVs and 2 to the class III HERVs. Many of these families have been identified previously, but six are described here for the first time and another four, for which only partial sequence information was previously available, were further characterized. Members of each of the 10 families are defective, and calculation of their integration dates suggested that most of them are likely to have been present within the human lineage since it diverged from the Old World monkeys more than 25 million years ago.

Oxidative DNA damage through long-range electron transfer

Nature ◽  
1996 ◽  
Vol 382 (6593) ◽  
pp. 731-735 ◽  
Author(s):  
Daniel B. Hall ◽  
R. Erik Holmlin ◽  
Jacqueline K. Barton
Keyword(s):  
Dna Damage ◽  
Electron Transfer ◽  
Long Range ◽  
Oxidative Dna Damage

scholarly journals Long range electronic transport in DNA molecules deposited across a disconnected array of metallic nanoparticles

2012 ◽  
Vol 13 (9-10) ◽  
pp. 967-992 ◽  
Author(s):  
Alexei D. Chepelianskii ◽  
D. Klinov ◽  
A. Kasumov ◽  
S. Guéron ◽  
O. Pietrement ◽  
...  
Keyword(s):  
Long Range ◽  
Electronic Transport ◽  
Metallic Nanoparticles ◽  
Dna Molecules

Isolation of chromosome 21–specific yeast artificial chromosomes from a total human genome library

1992 ◽  
Vol 1 (3) ◽  
pp. 222-225 ◽  
Author(s):  
I.M. Chumakov ◽  
I. Le Gall ◽  
A. Billault ◽  
P. Ougen ◽  
P. Soularue ◽  
...  
Keyword(s):  
Human Genome ◽  
Chromosome 21 ◽  
Artificial Chromosomes ◽  
Genome Library ◽  
Yeast Artificial Chromosomes

scholarly journals Preferred end coordinates and somatic variants as signatures of circulating tumor DNA associated with hepatocellular carcinoma

2018 ◽  
Vol 115 (46) ◽  
pp. E10925-E10933 ◽  
Author(s):  
Peiyong Jiang ◽  
Kun Sun ◽  
Yu K. Tong ◽  
Suk Hang Cheng ◽  
Timothy H. T. Cheng ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Peripheral Circulation ◽  
Sequence Information ◽  
Sequencing Analysis ◽  
Plasma Samples ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tumor Dna

Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment.

scholarly journals Detection of structural variations in densely-labelled optical DNA barcodes: A hidden Markov model approach

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259670
Author(s):  
Albertas Dvirnas ◽  
Callum Stewart ◽  
Vilhelm Müller ◽  
Santosh Kumar Bikkarolla ◽  
Karolin Frykholm ◽  
...  
Keyword(s):  
Markov Model ◽  
Hidden Markov Model ◽  
Large Scale ◽  
Hidden Markov ◽  
Sequence Information ◽  
True Positive ◽  
Dna Barcodes ◽  
Structural Variations ◽  
Genomic Alterations ◽  
Data Set

Large-scale genomic alterations play an important role in disease, gene expression, and chromosome evolution. Optical DNA mapping (ODM), commonly categorized into sparsely-labelled ODM and densely-labelled ODM, provides sequence-specific continuous intensity profiles (DNA barcodes) along single DNA molecules and is a technique well-suited for detecting such alterations. For sparsely-labelled barcodes, the possibility to detect large genomic alterations has been investigated extensively, while densely-labelled barcodes have not received as much attention. In this work, we introduce HMMSV, a hidden Markov model (HMM) based algorithm for detecting structural variations (SVs) directly in densely-labelled barcodes without access to sequence information. We evaluate our approach using simulated data-sets with 5 different types of SVs, and combinations thereof, and demonstrate that the method reaches a true positive rate greater than 80% for randomly generated barcodes with single variations of size 25 kilobases (kb). Increasing the length of the SV further leads to larger true positive rates. For a real data-set with experimental barcodes on bacterial plasmids, we successfully detect matching barcode pairs and SVs without any particular assumption of the types of SVs present. Instead, our method effectively goes through all possible combinations of SVs. Since ODM works on length scales typically not reachable with other techniques, our methodology is a promising tool for identifying arbitrary combinations of genomic alterations.

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