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Genome-wide maps and functional peak analysis of ultrashort single-stranded cell-free DNA (uscfDNA) and mononucleosomal cell-free DNA (mncfDNA) in human plasma

ABSTRACT: We report the application of an uscfDNA-oriented sequencing pipeline profiling the cell-free DNA populations in plasma. The uscfDNA sequencing pipeline include two uscfDNA-optimized extraction methods (QiaM and SPRI) and one control extraction method (QiaC). Final libraries are made with a single-stranded library prepartion kit. We generate genome-wide maps revealing the presence of an uscfDNA population (25-99bp) in addition to the mncfDNA (100-250bp).

ORGANISM(S): Homo sapiens

PROVIDER: GSE202430 | GEO | 2022/05/10

REPOSITORIES: GEO

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Digestion treatment of uscfDNA extracted by SPRI Method

Project description:We report the application of an uscfDNA-oriented sequencing pipeline profiling the cell-free DNA populations in plasma. The uscfDNA sequencing pipeline include two uscfDNA-optimized extraction methods (QiaM and SPRI) and one control extraction method (QiaC). Final libraries are made with a single-stranded library prepartion kit. We generate genome-wide maps revealing the presence of an uscfDNA population (25-99bp) in addition to the mncfDNA (100-250bp).

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Digestion treatment of uscfDNA extracted by QiaM Method

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uscfDNA-oriented sequencing pipeline profiling the cell-free DNA populations in plasma

Project description:uscfDNA-oriented sequencing pipeline profiling the cell-free DNA populations in plasma

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Advantages of Single-Stranded DNA Over Double-Stranded DNA Library Preparation for Capturing Cell-Free Tumor DNA in Plasma.

Project description:BackgroundSingle-stranded DNA (ssDNA) libraries have been shown to enrich shorter and more degraded DNA fragments than double-stranded DNA (dsDNA) libraries.ObjectiveIn this study, we evaluated whether ssDNA libraries captured more circulating tumor DNA (ctDNA) in plasma cell-free DNA (cfDNA).MethodsWe prepared dsDNA, ssDNA and pure-ssDNA (capture the preexisting ssDNA) libraries using ten plasma cfDNA samples. After low-pass whole genome sequencing, we calculated a duplicate rate to estimate library complexity and compared the library insert sizes between the different library methods. Finally, we estimated the ctDNA content and plasma genomic abnormality (PGA) score, an indicator of ctDNA burden.Results27 libraries were prepared and sequenced from the ten cfDNA samples. The duplicate rate in the ssDNA and pure-ssDNA libraries was significantly lower than in the dsDNA libraries (p < 0.001 and p < 0.01, respectively). ctDNA content and PGA scores were consistently higher in the ssDNA and pure-ssDNA libraries than in the matched dsDNA libraries (p < 0.005). The higher ctDNA content in ssDNA libraries was associated with smaller library insert size.ConclusionsssDNA libraries preserve more diversity and capture more ctDNA than dsDNA libraries. The ssDNA library method is preferred when performing genomic analysis of ctDNA.

| S-EPMC7216418 | biostudies-literature

Plasma contains ultrashort single-stranded DNA in addition to nucleosomal cell-free DNA.

Project description:Plasma cell-free DNA is being widely explored as a biomarker for clinical screening. Currently, methods are optimized for the extraction and detection of double-stranded mononucleosomal cell-free DNA of ∼160bp in length. We introduce uscfDNA-seq, a single-stranded cell-free DNA next-generation sequencing pipeline, which bypasses previous limitations to reveal a population of ultrashort single-stranded cell-free DNA in human plasma. This species has a modal size of 50nt and is distinctly separate from mononucleosomal cell-free DNA. Treatment with single-stranded and double-stranded specific nucleases suggests that ultrashort cell-free DNA is primarily single-stranded. It is distributed evenly across chromosomes and has a similar distribution profile over functional elements as the genome, albeit with an enrichment over promoters, exons, and introns, which may be suggestive of a terminal state of genome degradation. The examination of this cfDNA species could reveal new features of cell death pathways or it can be used for cell-free DNA biomarker discovery.

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Single-stranded Pre-methylated 5mC Adapters Uncover the Methylation Profile of Plasma Ultrashort Single-stranded Cell-free DNA

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

Project description:Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, 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 ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10(-5), Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10(-5)) and microbial cfDNA (71.3x, p 10(-5)). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods.

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Distinct Features of Plasma Ultrashort Single-Stranded Cell-Free DNA as Biomarkers for Lung Cancer Detection.

Project description:BackgroundUsing broad range cell-free DNA sequencing (BRcfDNA-Seq), a nontargeted next-generation sequencing (NGS) methodology, we previously identified a novel class of approximately 50 nt ultrashort single-stranded cell-free DNA (uscfDNA) in plasma that is distinctly different from 167 bp mononucleosomal cell-free DNA (mncfDNA). We hypothesize that uscfDNA possesses characteristics that are useful for disease detection.MethodsUsing BRcfDNA-Seq, we examined both cfDNA populations in the plasma of 18 noncancer controls and 14 patients with late-stage nonsmall cell lung carcinoma (NSCLC). In comparison to mncfDNA, we assessed whether functional element (FE) peaks, fragmentomics, end-motifs, and G-Quadruplex (G-Quad) signatures could be useful features of uscfDNA for NSCLC determination.ResultsIn noncancer participants, compared to mncfDNA, uscfDNA fragments showed a 45.2-fold increased tendency to form FE peaks (enriched in promoter, intronic, and exonic regions), demonstrated a distinct end-motif-frequency profile, and presented with a 4.9-fold increase in G-Quad signatures. Within NSCLC participants, only the uscfDNA population had discoverable FE peak candidates. Additionally, uscfDNA showcased different end-motif-frequency candidates distinct from mncfDNA. Although both cfDNA populations showed increased fragmentation in NSCLC, the G-Quad signatures were more discriminatory in uscfDNA. Compilation of cfDNA features using principal component analysis revealed that the first 5 principal components of both cfDNA subtypes had a cumulative explained variance of >80%.ConclusionsThese observations indicate that the distinct biological processes of uscfDNA and that FE peaks, fragmentomics, end-motifs, and G-Quad signatures are uscfDNA features with promising biomarker potential. These findings further justify its exploration as a distinct class of biomarker to augment pre-existing liquid biopsy approaches.

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

Project description:BackgroundCell-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.ResultsWe 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.ConclusionsWe propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

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Genome-wide maps and functional peak analysis of ultrashort single-stranded cell-free DNA (uscfDNA) and mononucleosomal cell-free DNA (mncfDNA) in human plasma

Project description:Genome-wide maps and functional peak analysis of ultrashort single-stranded cell-free DNA (uscfDNA) and mononucleosomal cell-free DNA (mncfDNA) in human plasma

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