scholarly journals Somatic mutation landscapes at single-molecule resolution

2021 ◽  
Author(s):  
Stefanie V. Lensing ◽  
Peter Ellis ◽  
Federico Abascal ◽  
Iñigo Martincorena ◽  
Robert J. Osborne
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Error Rates ◽  
Dna Molecules ◽  
Base Pairs ◽  
Single Dna Molecules ◽  
Sequencing Library ◽  
Somatic Mutagenesis ◽  
Qpcr Quantification ◽  
Duplex Sequencing

Abstract Somatic mutations drive cancer development and may contribute to ageing and other diseases. Yet, the difficulty of detecting mutations present only in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. To overcome these limitations, we introduce nanorate sequencing (NanoSeq), a new duplex sequencing protocol with error rates <5 errors per billion base pairs in single DNA molecules from cell populations. The version of the protocol described here uses clean genome fragmentation with a restriction enzyme to prevent end-repair-associated errors and ddBTPs/dATPs during A-tailing to prevent nick extension. Both changes reduce the error rate of standard duplex sequencing protocols by preventing the fixation of DNA damage into both strands of DNA molecules during library preparation. We also use qPCR quantification of the library prior to amplification to optimise the complexity of the sequencing library given the desired sequencing coverage, maximising duplex coverage. The sample preparation protocol takes between 1 and 2 days, depending on the number of samples processed. The bioinformatic protocol is described in:https://github.com/cancerit/NanoSeqhttps://github.com/fa8sanger/NanoSeq_Paper_Code

scholarly journals Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping

2021 ◽  
Author(s):  
Tslil Gabrieli ◽  
Yael Michaeli ◽  
Sigal Avraham ◽  
Dmitry Torchinsky ◽  
Matyas Juhasz ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Molecule ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Dna Molecules ◽  
Base Pairs ◽  
Single Dna Molecules ◽  
Genome Maps ◽  
Genome Methylation ◽  
Genomic Regions

ABSTRACTDNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and among the most significant epigenetic modifications. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in the CpG context allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. For this method, a CpG methyltransferase was used to transfer an azide to cytosines from a synthetic S-adenosyl-l-methionine cofactor analog. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased two-fold by the addition of a nucleosidase that degrades the inactive by-product of the azide-cofactor after labeling, and prevents its inhibitory effect. We first used the method to determine the decline in global DNA methylation in chronic lymphocytic leukemia patients and then performed whole genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published methylation maps produced by bisulfite sequencing. Although mapping resolution is limited by optical detection to 500-1000 base pairs, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

scholarly journals Single-molecule long-read sequencing reveals the chromatin basis of gene expression

2019 ◽  
Author(s):  
Yunhao Wang ◽  
Anqi Wang ◽  
Zujun Liu ◽  
Andrew Thurman ◽  
Linda S. Powers ◽  
...  
Keyword(s):  
Single Cell ◽  
Long Range ◽  
Single Molecule ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Single Cell Level ◽  
Dna Molecules ◽  
Cell Level ◽  
Single Dna Molecules ◽  
Long Read

ABSTRACTGenome-wide chromatin accessibility and nucleosome occupancy profiles have been widely investigated, while the long-range dynamics remains poorly studied at the single-cell level. Here we present a new experimental approach MeSMLR-seq (methyltransferase treatment followed by single-molecule long-read sequencing) for long-range mapping of nucleosomes and chromatin accessibility at single DNA molecules, and thus achieve comprehensive-coverage characterization of the corresponding heterogeneity. We applied MeSMLR-seq to haploid yeast, where single DNA molecules represent single cells, and thus we could investigate the combinatorics of many (up to 356) nucleosomes at long range in single cells. We illustrated the differential organization principles of nucleosomes surrounding transcription start site for silently- and actively-transcribed genes, at the single-cell level and in the long-range scale. The heterogeneous patterns of chromatin statuses spanning multiple genes were phased. Together with single-cell RNA-seq data, we quantitatively revealed how chromatin accessibility correlated with gene transcription positively in a highly-heterogeneous scenario. Moreover, we quantified the openness of promoters and investigated the coupled chromatin changes of adjacent genes at single DNA molecules during transcription reprogramming.

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

The Analyst ◽  
2019 ◽  
Vol 144 (3) ◽  
pp. 921-927 ◽  
Author(s):  
Jihyun Park ◽  
Seonghyun Lee ◽  
Nabin Won ◽  
Eunji Shin ◽  
Soo-Hyun Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Physical Map ◽  
Dna Molecules ◽  
Single Dna Molecules

Two-color DNA physical map for efficient identification of single DNA molecules.

Counting Single DNA Molecules in a Capillary with Radial Focusing

2003 ◽  
Vol 56 (3) ◽  
pp. 149 ◽  
Author(s):  
Jinjian Zheng ◽  
Edward S. Yeung
Keyword(s):  
Single Molecule ◽  
Detection Efficiency ◽  
Signal To Noise Ratio ◽  
Sampling Rate ◽  
Ccd Camera ◽  
Counting Efficiency ◽  
Spherical Particles ◽  
Dna Molecules ◽  
Single Dna Molecules ◽  
Relative Standard

For single-molecule detection, usually a small detection volume of 10 pL or less is used to improve the signal-to-noise ratio. Detection of every molecule in a sample requires that the sample be driven through a well-defined volume to facilitate laser excitation. We report a novel approach to count single DNA molecules with nearly 100% efficiency. By applying an electric field across a 40 cm long, 75 × 75 µm2 square capillary together with hydrodynamic flow from cathode to anode, we were able to concentrate more than 95% of DNA molecules into a 10 µm region at the centre of the capillary. The YOYO-1 labelled λ-DNA molecules were imaged with an intensified CCD camera. We found that the single DNA molecule detection efficiency in a 10–17 M solution was 114 ± 21%. The mobility of the DNA molecules after radial focusing was relatively constant, with relative standard deviations ranging from 0.8% to 1.4%. This allowed us to match the sampling rate to the length of the detection window to maximize counting efficiency. Analysis of a 40.2 nL injected plug of 2 × 10–14 M λ-DNA gave a result of 492 ± 73 molecules, which agreed well with the estimated value of 484. This method should be generally useful for counting deformable molecules or non-spherical particles at extremely low concentrations.

Molecular microscopy of DNA for genome analysis: optical mapping of restriction digests

1995 ◽  
Vol 53 ◽  
pp. 50-51
Author(s):  
Edward J. Huff ◽  
Weiwen Cai ◽  
Xinghua Hu ◽  
John Huang ◽  
Junping Jing ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Analysis ◽  
Optical Mapping ◽  
Ccd Camera ◽  
Dna Molecules ◽  
Specific Sequence ◽  
Single Dna Molecules ◽  
Single Clone ◽  
Rapid Generation ◽  
Glass Surfaces

Optical microscopy of individual DNA molecules has been an interesting technique for the past 15 years, but until recently has not been useful for genome analysis. We have developed Optical Mapping an emerging single molecule approach for the rapid generation of ordered restriction maps. Many identical individual DNA molecules from a single clone are elongated and fixed onto derivatized glass surfaces, digested with a restriction enzyme which cuts the DNA wherever a specific sequence pattern is found, stained with YOYO, and imaged with a cooled CCD camera attached to an automated epi-fluorescence microscope. Images are automatically processed to correct for non-uniform illumination, remove background, locate the DNA fragments, reject objects which do not look like single DNA molecules, recognize which fragments originate from an original uncut molecule, and calculate the relative sizes of the fragments by apparent length and fluorescence intensity. Results from many molecules are combined by clustering to recognize a consistent cutting pattern. Molecules which match the pattern are averaged to improve the sizing accuracy.

Determination of haplotypes from single DNA molecules: a method for single-molecule barcoding

2007 ◽  
Vol 28 (9) ◽  
pp. 913-921 ◽  
Author(s):  
Ming Xiao ◽  
Matthew P. Gordon ◽  
Angie Phong ◽  
Connie Ha ◽  
Ting-Fung Chan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Molecules ◽  
Single Dna Molecules

VISUALIZATION EX SITU OF SINGLE DNA MOLECULES INCUBATION: A FIRST STEP FOR QUANTITATIVE ANALYSIS ON MULTI-SITE DEGRADATION AND ENZYMATIC KINETICS

2009 ◽  
Vol 16 (01) ◽  
pp. 79-85
Author(s):  
XINYAN WANG ◽  
HAIJUN YANG ◽  
HUABIN WANG ◽  
PENG WANG ◽  
HAI LI
Keyword(s):  
Single Molecule ◽  
Ex Situ ◽  
In Vitro Tests ◽  
Dna Molecules ◽  
Single Molecule Level ◽  
Single Dna Molecules ◽  
Enzymatic Kinetics ◽  
Afm Imaging ◽  
Molecular Combing

Herein, we showed a different approach to directly single-molecule level visualization of the degradation of DNA in vitro tests using DNase I incubation based on high-resolution AFM imaging ex situ with fine relocation nanotechnology. A method of nanomanipulation termed as "modified dynamic molecular combing" (MDMC) was used to pattern DNA samples for further degradation and enzymatic kinetics. This strategy is potentially able to quantitatively address the mechanical-induced kinetic profiles of multi-site degradation of individual DNA molecules with very stable tension and strong immobilization on a surface and discover the mechanochemistry.

Positioning Isolation and Biochemical Analysis of Single DNA Molecules Based on Nanomanipulation and Single-Molecule PCR

2004 ◽  
Vol 126 (36) ◽  
pp. 11136-11137 ◽  
Author(s):  
Jun-hong Lü ◽  
Hai-kuo Li ◽  
Hong-jie An ◽  
Guo-hua Wang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Biochemical Analysis ◽  
Dna Molecules ◽  
Single Dna Molecules
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