scholarly journals Short single-stranded DNA with putative non-canonical structures comprises a novel class of plasma cell-free DNA

2021 ◽  
Author(s):  
Osamu Hisano ◽  
Takashi Ito ◽  
Fumihito Miura
Keyword(s):  
Binding Sites ◽  
Open Chromatin ◽  
Cell Free Dna ◽  
Single Stranded Dna ◽  
Dna Structures ◽  
Free Dna ◽  
G Quadruplex ◽  
Labeling Method ◽  
Purification Protocol ◽  
Library Preparation Protocol

Cell-free DNA (cfDNA) in human blood is currently investigated as a minimally invasive, highly informative biomarker. Here, we aimed to investigate the existence of the shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in human plasma, present at a molar concentration comparable to that of the nucleosome-sized fragments. These short fragments cannot be recovered by widely used cfDNA isolation methods, and are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. We established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of peaks called with these short fragments. Hence, we propose a novel class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

scholarly journals Short single-stranded DNAs with putative non-canonical structures comprise a new class of plasma cell-free DNA

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Osamu Hisano ◽  
Takashi Ito ◽  
Fumihito Miura
Keyword(s):  
Extracellular Dna ◽  
Open Chromatin ◽  
Dna Fragments ◽  
Cell Free Dna ◽  
Dna Structures ◽  
New Class ◽  
Free Dna ◽  
G Quadruplex ◽  
Labeling Method ◽  
Library Preparation Protocol

Abstract Background Cell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations. Results We aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments. Conclusions We propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

scholarly journals Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sara Lago ◽  
Matteo Nadai ◽  
Filippo M. Cernilogar ◽  
Maryam Kazerani ◽  
Helena Domíniguez Moreno ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Specific Gene ◽  
Open Chromatin ◽  
Rna Seq ◽  
Transcript Levels ◽  
Promoter Sequences ◽  
G Quadruplex ◽  
Cell Type Specific ◽  
Folding State

AbstractCell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity. By G4-ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin. Comparing G4 presence, location and transcript levels in liposarcoma cells to available data on keratinocytes, we showed that the same promoter sequences of the same genes in the two cell lines had different G4-folding state: high transcript levels consistently associated with G4-folding. Transcription factors AP-1 and SP1, whose binding sites were the most significantly represented in G4-folded sequences, coimmunoprecipitated with their G4-folded promoters. Thus, G4s and their associated transcription factors cooperate to determine cell-specific transcriptional programs, making G4s to strongly emerge as new epigenetic regulators of the transcription machinery.

scholarly journals Orientation-aware plasma cell-free DNA fragmentation analysis in open chromatin regions informs tissue of origin

2019 ◽  
Vol 29 (3) ◽  
pp. 418-427 ◽  
Author(s):  
Kun Sun ◽  
Peiyong Jiang ◽  
Suk Hang Cheng ◽  
Timothy H.T. Cheng ◽  
John Wong ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Plasma Cell ◽  
Open Chromatin ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tissue Of Origin

scholarly journals Nuclease deficiencies alter plasma cell-free DNA methylation profiles

2021 ◽  
pp. gr.275426.121
Author(s):  
Diana Siao Cheng Han ◽  
Meng Ni ◽  
Rebecca Wing Yan Chan ◽  
Danny Ka Lok Wong ◽  
Linda T Hiraki ◽  
...  
Keyword(s):  
Fragment Size ◽  
Cpg Islands ◽  
Periodic Pattern ◽  
Open Chromatin ◽  
Cell Free Dna ◽  
Free Dna ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Deficient Mice

The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation was explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in Dnase1l3-deficient mice was significantly hypomethylated, while cfDNA in Dnase1-deficient mice was hypermethylated. The cfDNA hypomethylation in Dnase1l3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in Dnase1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends and coverage at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affected cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology and cfDNA fragmentation.

scholarly journals Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Philip Burnham ◽  
Min Seong Kim ◽  
Sean Agbor-Enoh ◽  
Helen Luikart ◽  
Hannah A. Valantine ◽  
...  
Keyword(s):  
Library Preparation ◽  
Cell Free Dna ◽  
Single Stranded Dna ◽  
Dna Library ◽  
Free Dna

scholarly journals Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

2015 ◽  
Author(s):  
Philip Burnham ◽  
Min Seong Kim ◽  
Sean Agbor-Enoh ◽  
Helen Luikart ◽  
Hannah A Valantine ◽  
...  
Keyword(s):  
Lung Transplant ◽  
Preparation Method ◽  
Fragmentation Pattern ◽  
Library Preparation ◽  
Transplant Recipients ◽  
Cell Free Dna ◽  
Single Stranded Dna ◽  
Free Dna ◽  
Lung Transplant Recipients ◽  
Library Preparation Method

Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of cfDNA in blood, can be readily adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparation, however, is insensitive to ultrashort, degraded and single-stranded cfDNA. Drawing inspiration from recent technical advances in ancient genome analyses, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that the ssDNA library preparation yields a greater portion of sub-100 bp DNA, as well as an increased relative abundance of human mitochondrial cfDNA (10.7x) and microbial cfDNA (71.3x). We report the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We furthermore report the first observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. We found that donor-specific mitochondrial cfDNA molecules are significantly shorter than those specific to the recipient. The higher yield of viral, microbial and fungal sequences that result from the single-stranded ligation approach reduces the cost and increase the sensitivity of cfDNA-based monitoring for infectious complications after transplantation. An ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short fragment nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods.

scholarly journals Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome

2020 ◽  
Author(s):  
Sara Lago ◽  
Filippo M. Cernilogar ◽  
Maryam Kazerani ◽  
Helena Domíniguez Moreno ◽  
Matteo Nadai ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Specific Gene ◽  
Open Chromatin ◽  
Rna Seq ◽  
Transcript Levels ◽  
Promoter Sequences ◽  
G Quadruplex ◽  
Cell Type Specific ◽  
Folding State

AbstractCell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization1-3. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity4,5. By G4 ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin. Comparing G4 presence, location and transcript levels in liposarcoma cells to available data on keratinocytes, we showed that the same promoter sequences of the same genes in the two cell lines had different G4-folding state: high transcript levels consistently associated with high G4-folding. Transcription factors AP-1 and SP1, whose binding sites were the most significantly represented in G4-folded sequences, coimmunoprecipitated with their G4-folded promoters. Thus G4s and their associated transcription factors cooperate to determine cell-specific transcriptional programs, making G4s strongly emerge as new epigenetic regulators of the transcription machinery.

scholarly journals Plasma Contains Ultra-short Single-stranded DNA in Addition to Nucleosomal cfDNA

2021 ◽  
Author(s):  
Jordan C Cheng ◽  
Marco Morselli ◽  
Wei-Lun Huang ◽  
You Jeong Heo ◽  
Thalyta Pinheiro-Ferreira ◽  
...  
Keyword(s):  
Plasma Cell ◽  
Biomarker Discovery ◽  
Nuclear Genome ◽  
Cell Free Dna ◽  
Single Stranded Dna ◽  
Diagnostic Screening ◽  
Free Dna ◽  
Modal Size

Plasma cell-free DNA is a widely used biomarker for diagnostic screening. We introduce uscfDNA-seq, a single-stranded cell-free DNA NGS pipeline, which bypasses previous limitations to reveal a novel population of ultrashort single-stranded cell-free DNA in plasma with a modal size of 50nt. This species of cfDNA aligns predominantly to the nuclear genome and could potentially be used for novel biomarker discovery.

scholarly journals OCRDetector: Accurately Detecting Open Chromatin Regions via Plasma Cell-Free DNA Sequencing Data

2021 ◽  
Vol 22 (11) ◽  
pp. 5802
Author(s):  
Jiayin Wang ◽  
Liubin Chen ◽  
Xuanping Zhang ◽  
Yao Tong ◽  
Tian Zheng
Keyword(s):  
Plasma Cell ◽  
Housekeeping Genes ◽  
Read Depth ◽  
Regulatory Elements ◽  
Open Chromatin ◽  
Sequencing Data ◽  
Bioinformatics Pipeline ◽  
Cell Free Dna ◽  
Sequencing Coverage ◽  
Free Dna

Open chromatin regions (OCRs) are special regions of the human genome that can be accessed by DNA regulatory elements. Several studies have reported that a series of OCRs are associated with mechanisms involved in human diseases, such as cancers. Identifying OCRs using ATAC-seq or DNase-seq is often expensive. It has become popular to detect OCRs from plasma cell-free DNA (cfDNA) sequencing data, because both the fragmentation modes of cfDNA and the sequencing coverage in OCRs are significantly different from those in other regions. However, it is a challenging computational problem to accurately detect OCRs from plasma cfDNA-seq data, as multiple factors—e.g., sequencing and mapping bias, insufficient read depth, etc.—often mislead the computational model. In this paper, we propose a novel bioinformatics pipeline, OCRDetector, for detecting OCRs from whole-genome cfDNA sequencing data. The pipeline calculates the window protection score (WPS) waveform and the cfDNA sequencing coverage. To validate the proposed pipeline, we compared the percentage overlap of our OCRs with those obtained by other methods. The experimental results show that 81% of the TSS regions of housekeeping genes are detected, and our results have obvious tissue specificity. In addition, the overlap percentage between our OCRs and the high-confidence OCRs obtained by ATAC-seq or DNase-seq is greater than 70%.

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