scholarly journals ATAC-seq identifies thousands of extrachromosomal circular DNA in cancers and cell lines

2019 ◽  
Author(s):  
Pankaj Kumar ◽  
Shashi Kiran ◽  
Shekhar Saha ◽  
Zhangli Su ◽  
Teressa Paulsen ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
High Throughput Sequencing ◽  
Rolling Circle Amplification ◽  
Inverse Pcr ◽  
Open Chromatin ◽  
Lower Grade ◽  
Circular Dna ◽  
Rolling Circle ◽  
Circular Dnas

AbstractExtrachromosomal circular DNAs (eccDNAs) lack centromeres and are not passed equally into daughter cells and thus provide a source of cell-cell heterogeneity in normal and tumor cells. Previously we and other groups purified circular DNA and linearized them by rolling circle amplification for paired end high throughput sequencing to identify eccDNA in cells and tissues. We hypothesized that many eccDNA will have more open chromatin and so will be susceptible to linearization and sequencing by tagmentation. Indeed, we find that ATAC-seq on cell lines and cancers, without any enrichment of circular DNA, identifies thousands of eccDNAs. The identified eccDNAs in cell lines were validated by inverse PCR on DNA that survives exonuclease digestion of linear DNA and by metaphase FISH. We demonstrate that ATAC-seq data generated in Lower Grade Gliomas (LGG) and Glioblastomas (GBM) identify eccDNAs, including one containing the well-known EGFR gene amplicon from chr7. Many of the eccDNAs are identified even before amplification of the locus is detected by genotyping arrays. Thus, standard ATAC-seq is a sensitive method to detect segments of DNA that are present as eccDNA in a subset of the tumor cells, ready to be further amplified under appropriate selection, as during therapy.

scholarly journals ATAC-seq identifies thousands of extrachromosomal circular DNA in cancer and cell lines

2020 ◽  
Vol 6 (20) ◽  
pp. eaba2489 ◽  
Author(s):  
Pankaj Kumar ◽  
Shashi Kiran ◽  
Shekhar Saha ◽  
Zhangli Su ◽  
Teressa Paulsen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inverse Pcr ◽  
Driver Genes ◽  
Circular Dna ◽  
Cancer Driver ◽  
Genome Wide ◽  
Number Variation ◽  
Circular Dnas ◽  
Tumor Types ◽  
Pan Cancer

Extrachromosomal circular DNAs (eccDNAs) are somatically mosaic and contribute to intercellular heterogeneity in normal and tumor cells. Because short eccDNAs are poorly chromatinized, we hypothesized that they are sequenced by tagmentation in ATAC-seq experiments without any enrichment of circular DNA. Indeed, ATAC-seq identified thousands of eccDNAs in cell lines that were validated by inverse PCR and by metaphase FISH. ATAC-seq in gliomas and glioblastomas identify hundreds of eccDNAs, including one containing the well-known EGFR gene amplicon from chr7. More than 18,000 eccDNAs, many carrying known cancer driver genes, are identified in a pan-cancer analysis of ATAC-seq libraries from 23 tumor types. Somatically mosaic eccDNAs are identified by ATAC-seq even before amplification is recognized by genome-wide copy number variation measurements. Thus, ATAC-seq is a sensitive method to detect eccDNA present in a tumor at the pre-amplification stage and can be used to predict resistance to therapy.

Mitigating the Non-Specific Amplification in RCA: Assessment of the Role of Ligation and Exonuclease Digestion in Circular DNA Preparation

2021 ◽  
Author(s):  
Vandana Kuttappan Nair ◽  
Chandrika Sharma ◽  
Mrittika Sengupta ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
False Positive ◽  
Rolling Circle Amplification ◽  
Circular Dna ◽  
Rolling Circle ◽  
Specific Amplification ◽  
Exonuclease Digestion ◽  
Sticky End

<b>Layman Summary: </b>Rolling circle amplification (RCA) is a popular and extensively used bioanalytical tool. Like any nucleic acid amplifications, non-specific amplification may occur in it and risk generating false positive readouts. The work described in the manuscript investigates non-specific amplification in RCA as a function of ligation and exonuclease digestion assays during the synthesis of circular DNA. In particular, it investigates and compares the role of three different ligation techniques, namely splint-padlock ligation, cohesive end (sticky end ligation), and self-annealing ligation. In addition, it also probes the role of single exonuclease vs dual exonuclease digestions. We employed real time fluorescence to quantify the effect of these factors. Finally, our work hypothesizes the possible origins of non-specific amplification in RCA.

scholarly journals Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease

Plant Disease ◽  
2020 ◽  
Vol 104 (11) ◽  
pp. 3010-3018 ◽  
Author(s):  
Yuanjian Qiu ◽  
Song Zhang ◽  
Haodong Yu ◽  
Zhiyou Xuan ◽  
Liu Yang ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Close Association ◽  
Phylogenetic Analyses ◽  
Rolling Circle Amplification ◽  
Field Investigation ◽  
Broussonetia Papyrifera ◽  
New Members ◽  
Rolling Circle ◽  
Mulberry Leaf ◽  
Leaf Curl

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

Highly Sensitive Electrochemical Biosensor for Circulating Tumor Cells Detection via Dual-Aptamer Capture and Rolling Circle Amplification Strategy

2019 ◽  
Vol 15 (7) ◽  
pp. 1568-1577 ◽  
Author(s):  
Yang Wang ◽  
Kai Chang ◽  
Cheng Yang ◽  
Shujing Li ◽  
Lixin Wang ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Cancer Metastasis ◽  
Electrochemical Biosensor ◽  
Rolling Circle Amplification ◽  
K562 Cells ◽  
Detection Sensitivity ◽  
Experimental Conditions ◽  
Simple Method ◽  
Rolling Circle

A fast and simple strategy for early detection of circulating tumor cells (CTCs) is urgently required because of cancer metastasis. In this work, we assembled an electrochemical biosensor by two aptamers that could form hairpin and specifically recognize K562 cells. The thiolated capture aptamer was fixed on the gold electrode surface. The detection aptamer was linked with a primer at 3 end which could trigger rolling circle amplification to prolong the sequence of aptamer. The dual-aptamer model was fabricated to improve the capture specificity and efficiency for K562 cells. The rolling circle amplification improved the detection sensitivity by inhibiting electron transfer of [Fe(CN)6]3–/4– which could be measured by differential pulse voltammetry. The detection limit of 25 cells mL–1 and linear ranges of 1 × 10 2 to 1 × 105 cells mL–1 were obtained under optimal experimental conditions. Our work exhibited a label-free and simple method for detecting CTCs using cell-specific aptasensor, showing an expected possibility for further CTCs-related study and clinical applications of this novel method.

scholarly journals Single-nucleus chromatin accessibility reveals intratumoral epigenetic heterogeneity in IDH1 mutant gliomas

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Ruslan Al-Ali ◽  
Katharina Bauer ◽  
Jong-Whi Park ◽  
Ruba Al Abdulla ◽  
Valentina Fermi ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Lower Grade ◽  
Dna Hypermethylation ◽  
Molecular Group ◽  
Idh Mutations ◽  
Single Nucleus ◽  
Non Coding Rnas

AbstractThe presence of genome-wide DNA hypermethylation is a hallmark of lower grade gliomas (LGG) with isocitrate dehydrogenase (IDH) mutations. Further molecular classification of IDH mutant gliomas is defined by the presence (IDHmut-codel) or absence (IDHmut-noncodel) of hemizygous codeletion of chromosome arms 1p and 19q. Despite the DNA hypermethylation seen in bulk tumors, intra-tumoral heterogeneity at the epigenetic level has not been thoroughly analyzed. To address this question, we performed the first epigenetic profiling of single cells in a cohort of 5 gliomas with IDH1 mutation using single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq). Using the Fluidigm HT IFC microfluidics platform, we generated chromatin accessibility maps from 336 individual nuclei, and identified variable promoter accessibility of non-coding RNAs in LGGs. Interestingly, local chromatin structures of several non-coding RNAs are significant factors that contribute to heterogeneity, and show increased promoter accessibility in IDHmut-noncodel samples. As an example for clinical significance of this result, we identify CYTOR as a poor prognosis factor in gliomas with IDH mutation. Open chromatin assay points to differential accessibility of non-coding RNAs as an important source of epigenetic heterogeneity within individual tumors and between molecular subgroups. Rare populations of nuclei that resemble either IDH mutant molecular group co-exist within IDHmut-noncodel and IDHmut-codel groups, and along with non-coding RNAs may be an important issue to consider for future studies, as they may help guide predict treatment response and relapse.A web-based explorer for the data is available at shiny.turcanlab.org.

Evidence of Rubus Yellow Net Virus Integration into the Red Raspberry Genome

2020 ◽  
Vol 160 (6) ◽  
pp. 329-334
Author(s):  
Alfredo Diaz-Lara ◽  
Nola J. Mosier ◽  
Kristian Stevens ◽  
Karen E. Keller ◽  
Robert R. Martin
Keyword(s):  
High Throughput Sequencing ◽  
Rolling Circle Amplification ◽  
Standard Test ◽  
Molecular Techniques ◽  
Test Method ◽  
Plant Genome ◽  
Red Raspberry ◽  
Rolling Circle ◽  
Certification Programs ◽  
Dnase Treatment

Rubus yellow net virus (RYNV) infects Rubus spp., causing a severe decline when present in mixed infections with other viruses. RYNV belongs to the family Caulimoviridae, also known as plant pararetroviruses, which can exist as episomal or integrated elements (endogenous). Most of integrated pararetroviruses are noninfectious; however, a few cases have been reported where they excised from the plant genome and formed infectious particles. Graft transmission onto indicator plants R. occidentalis “Munger” has been the standard test method for RYNV detection in certification programs. Previously, it was noticed that some RYNV PCR-positive plants did not induce symptoms on “Munger”, suggesting an integration event. In this study, bio-indexing and different molecular techniques were employed to differentiate between integrated and episomal RYNV sequences. Reverse transcription-PCR using RYNV-specific oligonucleotides after DNase treatment generated positive results for the virus in graft transmissible isolates (episomal) only. To confirm these results, rolling circle amplification on DNA preparations from the same samples resulted in amplicons identified as RYNV only from plants with graft transmissible RYNV. High-throughput sequencing was used to identify the RYNV-like sequences present in the host DNA. These results indicate the integration of RYNV into the red raspberry genome and highlight the necessity to recognize this phenomenon (integration) in future Rubus quarantine and certification programs.

Mitigating the Non-Specific Amplification in RCA: Assessment of the Role of Ligation and Exonuclease Digestion in Circular DNA Preparation

2021 ◽  
Author(s):  
Vandana Kuttappan Nair ◽  
Chandrika Sharma ◽  
Mrittika Sengupta ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
False Positive ◽  
Rolling Circle Amplification ◽  
Circular Dna ◽  
Rolling Circle ◽  
Specific Amplification ◽  
Exonuclease Digestion ◽  
Sticky End

<b>Layman Summary: </b>Rolling circle amplification (RCA) is a popular and extensively used bioanalytical tool. Like any nucleic acid amplifications, non-specific amplification may occur in it and risk generating false positive readouts. The work described in the manuscript investigates non-specific amplification in RCA as a function of ligation and exonuclease digestion assays during the synthesis of circular DNA. In particular, it investigates and compares the role of three different ligation techniques, namely splint-padlock ligation, cohesive end (sticky end ligation), and self-annealing ligation. In addition, it also probes the role of single exonuclease vs dual exonuclease digestions. We employed real time fluorescence to quantify the effect of these factors. Finally, our work hypothesizes the possible origins of non-specific amplification in RCA.

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