Ontology highlight
ABSTRACT:
PROVIDER: PRJNA488349 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7782693_1.fastq.gz | Fastqsanger.gz | |||
| SRR7782693_2.fastq.gz | Fastqsanger.gz | |||
| SRR7782694_1.fastq.gz | Fastqsanger.gz | |||
| SRR7782694_2.fastq.gz | Fastqsanger.gz |
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Project description:Human mitochondrial DNA (mtDNA) variations, particularly the presence of both mutated and wild type mtDNA known as mtDNA heteroplasmies, have garnered increasing attention due to their clinical relevance to numerous diseases. Nonetheless, the absence of a feasible and economical approach has hampered large-scale population studies on mtDNA variations. Here, we present a novel human mtDNA sequencing method called 123-seq, which is based on the 123-multiplex PCR and completed in consecutive three-step biochemical reactions, with experimental validation of its reliability. The entire library construction process takes only about 90 minutes, enabling efficient capture, amplification, and library construction of mtDNA at the bulk genomic DNA level or single-cell level in a streamlined workflow. We then applied our method to assess single-cell mtDNA mutations in a human sample, profiling over 286 T lymphocytes from a 79-year-old female. We discovered that over 90% of cells carried at least one mtDNA mutation with variant allele frequencies (VAFs) exceeding 20%. Furthermore, 83.78% of these mutation sites were enriched in the protein-coding regions of mtDNA. Our findings suggest that mtDNA mutations with functional significance may be widespread in advanced age, indicating a potential link to T cell aging. This emphasizes the need for further investigation into the dynamics of age-related mtDNA mutations at the single-cell level.
2024-06-30 | GSE266764 | GEO
Project description:Background: Cell free DNA (cfDNA) in plasma has received increasing attention and has been studied in a broad range of clinical conditions implicating inflammation, cancer, and aging. However, few studies have focused on mitochondrial DNA (mtDNA) in the cell free form. This study characterized the size distribution and sequence characteristics of plasma cell free mtDNA (cf mtDNA) in humans.Methods and Results: We optimized DNA isolation and next-generation sequencing library preparation protocols to better retain short DNA fragments from plasma, and applied these optimized methods to plasma samples from patients with sepsis. After massive parallel sequencing, we verified that our methods can retain substantially shorter DNA fragments than the standard isolation method, resulting in an average of 11.5 fold increase in short DNA fragments yield (DNA < 100bp). We report that cf mtDNA in plasma is highly enriched in short-size cfDNA (30 ~ 60 bp), which is much shorter than the value previously reported (~140 bp). Motivated by this unique size distribution, we size-selected short cfDNA fragments from the sequencing library, which further increased the mtDNA recovery rate by an average of 10.4 fold. Using this approach we detected mixtures of different mtDNA sequences, termed heteroplasmy, in plasma from 3 patients. In one patient who previously received bone marrow transplantation, different minor allele frequencies were observed between plasma and white blood cells (WBC) at heteroplasmic mtDNA sites, consistent with mixed-tissue origin for plasma DNA.Conclusion: mtDNA in plasma exists as very short fragments that exhibit mtDNA heteroplasmy distribution differences from that found in a single organ/tissue. This study is the first report of genome wide identification of mtDNA heteroplasmy in human plasma. Our optimized method can be used to investigate the potential utility of cf mtDNA fragments and heteroplasmy as biomarkers in various diseases.
2016-05-07 | GSE81178 | GEO
Project description:Developing a single-cell multi-omics technology with mtDNA mutation profiling at its core will enable precise determination of the relationship between mtDNA mutations and cellular phenotypes. To date, the development of permeability-engineered compartmentalization for the isolation and single-cell library preparation of individual cells, which includes Tn5 tagmentation and single-cell barcoding operations in the permeabilized microcapsule format, remains a formidable challenge. Herein, we present a approach for single-cell mitochondrial analysis, harnessing microfluidics to generate heterogeneous hydrogels as artificial membranes. These hydrogels serve to regulate the passage of mitochondrial DNA and nuclear genomes subsequent to the cell lysis step. This approach enables high-throughput mitochondrial DNA genotyping and accessible chromatin profiling at the single-cell level, significantly enhancing the capabilities of permeability-engineered compartmentalization.We conducted a comprehensive evaluation of cell retention and the selective permeability of the capsule reaction system, optimizing conditions for cell lysis, Tn5 tagmentation, and barcode labeling. Validation experiments were performed using Human 293T and Mouse 3T3 cells to assess identification and quantification performance. Our method offers a new strategy for the study of the functions of various mtDNA mutations in biological processes, while also offering a new system for the construction of single-cell multi-omics libraries.
2023-10-10 | GSE244725 | GEO
Project description:In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1-3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA. Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA (>10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability.
2023-12-20 | GSE246469 | GEO
Project description:This dataset contains sequencing data from a large-scale study of mtDNA variations measured, using a sensitive mtDNA-targeted sequencing method called STAMP, in lymphoblast and blood samples of Huntington’s Disease patients.
| EGAD00001005723 | EGA
Project description:Maternal inheritance of mitochondrial DNA (mtDNA) is highly conserved in metazoans. While many species eliminate paternal mtDNA during late sperm development to foster maternal inheritance, the regulatory mechanisms governing this process remain elusive. Through a large-scale genetic screen in Drosophila, we identified 47 mutant lines exhibiting substantial retention of mtDNA in mature sperm. We mapped one line to Poldip2, a gene predominantly expressed in the testis. Disruption of Poldip2 led to pronounced mtDNA retention in mature sperm and subsequent paternal transmission to progeny. Further investigation via imaging, biochemical analyses and ChIP assays revealed that POLDIP2 is a mitochondrial matrix protein capable of binding to mtDNA. Moreover, we uncovered that CLPX, a key component of the major mitochondrial protease, binds to POLDIP2 to co-regulate mtDNA elimination in Drosophila spermatids. This study shed light on the mechanisms underlying mtDNA removal during spermatogenesis, underscoring the pivotal role of this process in safeguarding maternal inheritance.
2024-06-04 | E-MTAB-14100 | biostudies-arrayexpress
Project description:Background: Developments in DNA resequencing microarrays include mitochondrial DNA (mtDNA) sequencing and mutation detection. During automated analysis to sequence mtDNA, bases can be identified as wildtype, variant, or there may be a failure to assign a base (N-call) when compared to the mtDNA reference sequence (rCRS). The purpose of our study was to re-examine the N-call distribution of mtDNA samples, based on the hypothesis that N-calls may represent insertions or deletions in mtDNA. Findings: We analysed mtDNA from 16 patient samples (8 blood/bone marrow origin and 8 from cultured fibroblasts) using the Affymetrix MitoChip v.2.0 which has 2 probe areas, one for sequencing compared to the rCRS and one for 500 common haplotypes. N-calls by the proprietary GSEQ software were significantly reduced when the freeware on-line algorithm M-bM-^@M-^XsPROFILERM-bM-^@M-^Y was utilized. Interestingly, this decrease in N-calls, in both sections of the MitoChip, had no effect on the homoplasmic or heteroplasmic mutation levels compared to GSEQ software. We then used conventional DNA sequencing to analyse continuous N-call stretches identified by sPROFILER, ranging from 2 to 22 bases, to identify changes resulting in base calling failures. Conclusions: Our study is the first to analyse N-calls produced from GSEQ software for the MitoChip v2.0. We found that by narrowing the focus to stretches of N-calls revealed by sPROFILER, conventional sequencing was able to identify unique deletions and insertions. This study also appeared to support the contention that the GSEQ is more capable of assigning bases when used in conjunction with sPROFILER. mtDNA from 16 patient samples were analysed using Mitochip v2.0. Eight samples were DNA extractions directly from blood/bone marrow. The other eight samples were DNA extractions from primary cultured fibroblasts. Results were generated in GSEQ and reanalysed in sPROFILER for comparison.
2012-05-17 | E-GEOD-29550 | biostudies-arrayexpress
Project description:Expression data for validation of single cell cDNA amplification method (V1V3 method)
| PRJNA104647 | ENA
Project description:Validation of XACTLY method on fragmented human gDNA
| PRJNA579700 | ENA