KAS-seq: genome-wide sequencing of single-stranded DNA by N3-kethoxal–assisted labeling

Lyu, Ruitu; Wu, Tong; Zhu, Allen; West-Szymanski, Diana C.; Weng, Xiaocheng; Chen, Meng-Jie; He, Chuan

doi:10.1038/s41596-021-00647-6

Cited by 22 publications

(29 citation statements)

References 47 publications

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“…In summary, our results show that spKAS-seq can be used to generate a comprehensive map of genome-wide R-loops with high resolution and sensitivity [38][39][40][41][42]. [11,12]. KAS-pipe2 supports differential RNA Pols activity analysis for time-course KAS-seq data.…”

Section: Genome-wide R-loop Identification Using Spkas-seq Datamentioning

confidence: 63%

“…Time-course KAS-seq experiments can provide insights into the regulatory mechanisms of gene expression by capturing transient transcriptional dynamics with temporal resolution[11, 12]. KAS-pipe2 supports differential RNA Pols activity analysis for time-course KAS-seq data.…”

Section: Resultsmentioning

confidence: 99%

“…Compared to the previous version of the pipeline (KAS-pipe), which only includes a few standalone shell scripts [12], KAS-pipe2 adopts a new framework design to implement functionalities under the usage: KAS-pipe2 <subcommand> [options], which is convenient to update with new features and has been adopted in many other popular packages, such as deeptools and samtools. KAS-pipe2 not only provides foundational tools for analysis of KAS-seq and spKAS-seq data, but it also includes many expanded features to facilitate the data interpretation.…”

Section: Discussionmentioning

confidence: 99%

“…The experimental procedures of N 3 -kethoxal labeling and genomic DNA fragmentation are exactly the same for KAS-seq and spKAS-seq experiments, which have been described previously [11,12]. In brief, after the completion of gDNA N 3 -kethoxal labeling and fragmentation, 5% of the sonicated DNA was reserved as input and the remaining 95% was used for enrichment with 10 µL Dynabeads MyOne Streptavidin C1 (Thermo, 65001).…”

Section: Kas-seq and Spkas-seq Experimentsmentioning

confidence: 99%

“…To overcome these issues, we have developed kethoxal-assisted ssDNA sequencing (KAS-seq), a technology that can sensitively detect ‘transcription bubble’, the short stretches of unwound DNA that form when RNA Pols engage DNA for transcription. We have shown that the transient ssDNA present in transcription bubbles provides a more direct readout of transcriptional activity in situ than the nascent RNAs themselves[11, 12]. With the rapid (within 5 min), sensitive, and specific chemical reactions between N 3 -kethoxal and guanine bases in ssDNA, KAS-seq can work with as few as 1,000 cells or even with frozen tissues (Fig.…”

Section: Introductionmentioning

confidence: 99%

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KAS-pipe2: a flexible toolkit for exploring KAS-seq and spKAS-seq data

Lyu

Park

et al. 2022

Preprint

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Kethoxal-assisted ssDNA sequencing (KAS-seq) is gaining popularity as a robust and effective approach to study the dynamics of transcriptionally engaged RNA polymerases through profiling of genome-wide single-stranded DNA (ssDNA). Its latest variant, spKAS-seq, a strand-specific version of KAS-seq, has been developed to map genome-wide R-loop structures by detecting imbalances of ssDNA on two strands. However, user-friendly, open-source analysis pipelines for KAS-seq data are still lacking. Here we present KAS-pipe2 as a flexible and integrated toolkit to facilitate the analysis and interpretation of KAS-seq data. KAS-pipe2 can perform standard analyses such as quality control, read alignment, and differential RNA polymerase activity analysis. In addition, KAS-pipe2 introduces many novel features, including, but not limited to: calculation of transcriptional indexes, identification of single-stranded transcribing enhancers, and high-resolution mapping of R-loops. We use benchmark datasets to demonstrate that KAS-pipe2 provides a powerful framework to study transient transcriptional regulatory programs. KAS-pipe2 is available at https://github.com/Ruitulyu/KAS-pipe2.

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Section: Genome-wide R-loop Identification Using Spkas-seq Datamentioning

confidence: 63%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Kas-seq and Spkas-seq Experimentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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KAS-pipe2: a flexible toolkit for exploring KAS-seq and spKAS-seq data

Lyu

Park

et al. 2022

Preprint

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Adrenergic and mesenchymal signatures are identifiable in cell‐free DNA and correlate with metastatic disease burden in children with neuroblastoma

Vayani,

Kaufman,

Moore

et al. 2023

Pediatric Blood & Cancer

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BackgroundCell‐free DNA (cfDNA) profiles of 5‐hydroxymethylcytosine (5‐hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES‐specific signatures could be quantified in cfDNA 5‐hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma.MethodsWe previously performed an integrative analysis to identify ADRN and MES‐specific genes (n = 373 and n = 159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high‐risk patients including 21 patients with serial samples.ResultsSamples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures (p < .001). While ADRN and MES signature scores tracked together in serially collected samples, we identified instances of patients with increases in either MES or ADRN score at relapse.ConclusionsWhile it is feasible to identify ADRN and MES signatures using 5‐hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing.

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Exploring transient global transcriptional changes induced by ascorbic acid revealed via atKAS-seq profiling

Liu,

He,

2024

Funct Integr Genomics

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KAS-seq: genome-wide sequencing of single-stranded DNA by N3-kethoxal–assisted labeling

Cited by 22 publications

References 47 publications

KAS-pipe2: a flexible toolkit for exploring KAS-seq and spKAS-seq data

KAS-pipe2: a flexible toolkit for exploring KAS-seq and spKAS-seq data

Adrenergic and mesenchymal signatures are identifiable in cell‐free DNA and correlate with metastatic disease burden in children with neuroblastoma

Exploring transient global transcriptional changes induced by ascorbic acid revealed via atKAS-seq profiling

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