Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

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Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

10.1101/118786 ◽

2017 ◽

Author(s):

Jason C. Bell ◽

David Jukam ◽

Nicole A. Teran ◽

Viviana I. Risca ◽

Owen K. Smith ◽

...

Keyword(s):

Rna Sequencing ◽

Dosage Compensation ◽

Rna Processing ◽

De Novo ◽

Critical Component ◽

Proximity Ligation ◽

Ribonucleoprotein Complexes ◽

Genome Wide ◽

Sequencing Method ◽

Nascent Transcripts

AbstractRNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

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A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures

Nucleic Acids Research ◽

10.1093/nar/gkz731 ◽

2019 ◽

Vol 47 (20) ◽

pp. e125-e125 ◽

Cited By ~ 8

Author(s):

Ning Zhang ◽

Shundi Shi ◽

Tony Z Jia ◽

Ashley Ziegler ◽

Barney Yoo ◽

...

Keyword(s):

Rna Sequencing ◽

De Novo ◽

Direct Analysis ◽

Complete Sequence ◽

Complete Understanding ◽

Sequencing Method ◽

Single Base Resolution ◽

Base Modifications ◽

Real Complex ◽

Usage Efficiency

Abstract A complete understanding of the structural and functional potential of RNA requires understanding of chemical modifications and non-canonical bases; this in turn requires advances in current sequencing methods to be able to sequence not only canonical ribonucleotides, but at the same time directly sequence these non-standard moieties. Here, we present the first direct and modification type-independent RNA sequencing method via introduction of a 2-dimensional hydrophobic end-labeling strategy into traditional mass spectrometry-based sequencing (2D HELS MS Seq) to allow de novo sequencing of RNA mixtures and enhance sample usage efficiency. Our method can directly read out the complete sequence, while identifying, locating, and quantifying base modifications accurately in both single and mixed RNA samples containing multiple different modifications at single-base resolution. Our method can also quantify stoichiometry/percentage of modified RNA versus its canonical counterpart RNA, simulating a real biological sample where modifications exist but may not be 100% at a particular site in the RNA. This method is a critical step towards fully sequencing real complex cellular RNA samples of any type and containing any modification type and can also be used in the quality control of modified therapeutic RNAs.

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Efficient de novo assembly of eleven human genomes using PromethION sequencing and a novel nanopore toolkit

10.1101/715722 ◽

2019 ◽

Cited By ~ 21

Author(s):

Kishwar Shafin ◽

Trevor Pesout ◽

Ryan Lorig-Roach ◽

Marina Haukness ◽

Hugh E. Olsen ◽

...

Keyword(s):

Human Genome ◽

De Novo ◽

Proximity Ligation ◽

Current State ◽

Human Genomes ◽

Sequencing Method ◽

Human Genome Assembly ◽

Long Read ◽

Genome Assemblies ◽

Assembly Performance

AbstractPresent workflows for producing human genome assemblies from long-read technologies have cost and production time bottlenecks that prohibit efficient scaling to large cohorts. We demonstrate an optimized PromethION nanopore sequencing method for eleven human genomes. The sequencing, performed on one machine in nine days, achieved an average 63x coverage, 42 Kb read N50, 90% median read identity and 6.5x coverage in 100 Kb+ reads using just three flow cells per sample. To assemble these data we introduce new computational tools: Shasta - a de novo long read assembler, and MarginPolish & HELEN - a suite of nanopore assembly polishing algorithms. On a single commercial compute node Shasta can produce a complete human genome assembly in under six hours, and MarginPolish & HELEN can polish the result in just over a day, achieving 99.9% identity (QV30) for haploid samples from nanopore reads alone. We evaluate assembly performance for diploid, haploid and trio-binned human samples in terms of accuracy, cost, and time and demonstrate improvements relative to current state-of-the-art methods in all areas. We further show that addition of proximity ligation (Hi-C) sequencing yields near chromosome-level scaffolds for all eleven genomes.

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A general LC/MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures

10.1101/643387 ◽

2019 ◽

Author(s):

Ning Zhang ◽

Shundi Shi ◽

Tony Z. Jia ◽

Ashley Ziegler ◽

Barney Yoo ◽

...

Keyword(s):

Rna Sequencing ◽

De Novo ◽

Direct Analysis ◽

Complete Sequence ◽

Complete Understanding ◽

Sequencing Method ◽

Single Base Resolution ◽

Base Modifications ◽

Real Complex ◽

Usage Efficiency

ABSTRACTA complete understanding of the structural and functional potential of RNA requires understanding of chemical modifications and noncanonical bases; this in turn requires advances in current sequencing methods to be able to sequence not only canonical ribonucleotides, but at the same time directly sequence these nonstandard moieties. Here, we present the first direct and modification type-independent RNA sequencing method via integration of a hydrophobic end-labeling strategy with of 2-D mass-retention time LC/MS analysis to allow de novo sequencing of RNA mixtures and enhance sample usage efficiency. Our method can directly read out the complete sequence, while identifying, locating, and quantifying base modifications accurately in both single and mixed RNA samples containing multiple different modifications at single-base resolution. Our method can also quantify stoichiometry/percentage of modified RNA vs. its canonical counterpart RNA, simulating a real biological sample where modifications exist but may not be 100% at a particular site of the RNA. This method is a critical step towards fully sequencing real complex cellular RNA samples of any type and containing any modification types and can also be used in the quality control of modified therapeutic RNAs.

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Analysis of RNA processing and m6A modification using nanopore direct RNA sequencing.

10.26226/morressier.5ebd45acffea6f735881af26 ◽

2020 ◽

Author(s):

Matthew Parker

Keyword(s):

Rna Sequencing ◽

Rna Processing

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Faculty Opinions recommendation of Genome-wide profiling of heritable and de novo STR variations.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727556430.793546826 ◽

2018 ◽

Author(s):

Melanie Bahlo

Keyword(s):

De Novo ◽

Genome Wide

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Genome-wide correlation analysis to identify amplitude regulators of circadian transcriptome output

Scientific Reports ◽

10.1038/s41598-020-78851-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Evan S. Littleton ◽

Madison L. Childress ◽

Michaela L. Gosting ◽

Ayana N. Jackson ◽

Shihoko Kojima

Keyword(s):

Clock Genes ◽

Circadian System ◽

Average Level ◽

Relative Amplitude ◽

Critical Component ◽

Period 2 ◽

Genome Wide ◽

Mouse Tissues ◽

Rhythmic Gene ◽

Insight Into

AbstractCell-autonomous circadian system, consisting of core clock genes, generates near 24-h rhythms and regulates the downstream rhythmic gene expression. While it has become clear that the percentage of rhythmic genes varies among mouse tissues, it remains unclear how this variation can be generated, particularly when the clock machinery is nearly identical in all tissues. In this study, we sought to characterize circadian transcriptome datasets that are publicly available and identify the critical component(s) involved in creating this variation. We found that the relative amplitude of 13 genes and the average level of 197 genes correlated with the percentage of cycling genes. Of those, the correlation of Rorc in both relative amplitude and the average level was one of the strongest. In addition, the level of Per2AS, a novel non-coding transcript that is expressed at the Period 2 locus, was also linearly correlated, although with a much lesser degree compared to Rorc. Overall, our study provides insight into how the variation in the percentage of clock-controlled genes can be generated in mouse tissues and suggests that Rorc and potentially Per2AS are involved in regulating the amplitude of circadian transcriptome output.

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Genome-wide methylation sequencing identifies progression-related epigenetic drivers in myelodysplastic syndromes

Cell Death and Disease ◽

10.1038/s41419-020-03213-2 ◽

2020 ◽

Vol 11 (11) ◽

Author(s):

Jing-dong Zhou ◽

Ting-juan Zhang ◽

Zi-jun Xu ◽

Zhao-qun Deng ◽

Yu Gu ◽

...

Keyword(s):

Cancer Progression ◽

Myelodysplastic Syndromes ◽

Bisulfite Sequencing ◽

De Novo ◽

Dna Hypermethylation ◽

Reduced Representation ◽

Targeted Bisulfite Sequencing ◽

Specific Pcr ◽

Genome Wide ◽

Potential Biomarker

AbstractThe potential mechanism of myelodysplastic syndromes (MDS) progressing to acute myeloid leukemia (AML) remains poorly elucidated. It has been proved that epigenetic alterations play crucial roles in the pathogenesis of cancer progression including MDS. However, fewer studies explored the whole-genome methylation alterations during MDS progression. Reduced representation bisulfite sequencing was conducted in four paired MDS/secondary AML (MDS/sAML) patients and intended to explore the underlying methylation-associated epigenetic drivers in MDS progression. In four paired MDS/sAML patients, cases at sAML stage exhibited significantly increased methylation level as compared with the matched MDS stage. A total of 1090 differentially methylated fragments (DMFs) (441 hypermethylated and 649 hypomethylated) were identified involving in MDS pathogenesis, whereas 103 DMFs (96 hypermethylated and 7 hypomethylated) were involved in MDS progression. Targeted bisulfite sequencing further identified that aberrant GFRA1, IRX1, NPY, and ZNF300 methylation were frequent events in an additional group of de novo MDS and AML patients, of which only ZNF300 methylation was associated with ZNF300 expression. Subsequently, ZNF300 hypermethylation in larger cohorts of de novo MDS and AML patients was confirmed by real-time quantitative methylation-specific PCR. It was illustrated that ZNF300 methylation could act as a potential biomarker for the diagnosis and prognosis in MDS and AML patients. Functional experiments demonstrated the anti-proliferative and pro-apoptotic role of ZNF300 overexpression in MDS-derived AML cell-line SKM-1. Collectively, genome-wide DNA hypermethylation were frequent events during MDS progression. Among these changes, ZNF300 methylation, a regulator of ZNF300 expression, acted as an epigenetic driver in MDS progression. These findings provided a theoretical basis for the usage of demethylation drugs in MDS patients against disease progression.

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