Mining for viral fragments in methylation enriched sequencing data
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR579156_1.fastq.gz | Fastqsanger.gz | |||
| ERR579156_2.fastq.gz | Fastqsanger.gz | |||
| ERR579157_1.fastq.gz | Fastqsanger.gz | |||
| ERR579157_2.fastq.gz | Fastqsanger.gz | |||
| ERR579158_1.fastq.gz | Fastqsanger.gz |
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Project description:Most next generation sequencing experiments generate more data than is usable for the experimental set up. For example, methyl-CpG binding domain (MBD) affinity purification based sequencing is often used for DNA-methylation profiling, but up to 30% of the sequenced fragments cannot be mapped uniquely to the reference genome. Here we present and evaluate a methodology for the identification of viruses in these otherwise unused paired-end MBD-seq data. Viral detection is accomplished by mapping non-reference alignable reads to a comprehensive set of viral genomes. As viruses play an important role in epigenetics and cancer development, 92 (pre)malignant and benign samples, originating from two different collections of cervical samples and related cell lines, were used in this study. These samples include primary carcinomas (n = 22), low- and high-grade cervical intraepithelial neoplasia (CIN1 and CIN2/3 - n = 2/n = 30) and normal tissue (n = 20), as well as control samples (n = 17). Viruses that were detected include phages, adenoviruses, herpesviridae and HPV. HPV, which causes virtually all cervical cancers, was identified in 95% of the carcinomas, 100% of the CIN2/3 samples, both CIN1 samples and in 55% of the normal samples. Comparing the amount of mapped fragments on HPV for each HPV-infected sample yielded a significant difference between normal samples and carcinomas or CIN2/3 samples (adjusted p-values resp. <10(-5), <10(-5)), reflecting different viral loads and/or methylation degrees in non-normal samples. Fragments originating from different HPV types could be distinguished and were independently validated by PCR-based assays in 71% of the detections. In conclusion, although limited by the a priori knowledge of viral reference genome sequences, the proposed methodology can provide a first confined but substantial insight into the presence, concentration and types of methylated viral sequences in MBD-seq data at low additional cost.
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Project description:We investigated the genome-wide distribution of Okazaki fragments in the commonly used laboratory Saccharomyces cerevisiae strain S288C to study the DNA replication model adopted by the budding yeast. The method based upon lambda exonuclease digestion for purification of RNA-primed replication intermediates was first improved to be suitable for the purification of Okazaki fragments. Then, we used this improved method to purify Okazaki fragments from S288C yeast cells, followed by Illumina sequencing. We found that the expected asymmetric distribution of Okazaki fragments around confirmed replication origins, which was derived from the semi-discontinuous DNA replication model, was not observed on S. cerevisiae chromosomes. Even around two highly efficient replication origins, ARS522 and ARS416, the ratios of Okazaki fragments on both strands were inconsistent with the semi-discontinuous DNA replication model. Our study supported the discontinuous DNA replication model. Besides, we also observed that Okazaki fragments were overpresented in the transcribed regions in S. cerevisiae mitochondrial genome, which indicated the interplay between transcription and DNA replication.
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Project description:We report a modulated expression of tRNA fragments and microRNAs linked to chondrosarcoma progression and demonstrate a method for re-modulating this pathway
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Project description:We investigated the genome-wide distribution of Okazaki fragments in the commonly used laboratory Saccharomyces cerevisiae strain S288C to study the DNA replication model adopted by the budding yeast. The method based upon lambda exonuclease digestion for purification of RNA-primed replication intermediates was first improved to be suitable for the purification of Okazaki fragments. Then, we used this improved method to purify Okazaki fragments from S288C yeast cells, followed by Illumina sequencing. We found that the expected asymmetric distribution of Okazaki fragments around confirmed replication origins, which was derived from the semi-discontinuous DNA replication model, was not observed on S. cerevisiae chromosomes. Even around two highly efficient replication origins, ARS522 and ARS416, the ratios of Okazaki fragments on both strands were inconsistent with the semi-discontinuous DNA replication model. Our study supported the discontinuous DNA replication model. Besides, we also observed that Okazaki fragments were overpresented in the transcribed regions in S. cerevisiae mitochondrial genome, which indicated the interplay between transcription and DNA replication. Examination of the distribution of Okazaki fragments in Saccharomyces cerevisiae strain S288C.
2013-06-18 | E-GEOD-42929 | biostudies-arrayexpress
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.
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Project description:We used 2', 3'-cyclic phosphate cDNA synthesis and Illumina sequencing to identify and quantify metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs. To make cDNA libraries, we exploited the 2’, 3’-cyclic phosphate at the end of RNA fragments produced by RNase L and other metal-ion-independent endoribonucleases. Using Arabidopsis thaliana tRNA ligase, RNA fragments with 2’, 3’-cyclic phosphates were covalently attached to defined RNA linkers containing an 8-base long unique molecular identifier (UMI) sequence. Libraries prepared in this manner contain cDNA derived exclusively from RNA fragments with 2', 3'-cyclic phosphates. The UMI sequence allows for detailed quantitation. We optimized and validated 2’, 3’-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A, and RNA from uninfected and poliovirus-infected HeLa cells.
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Project description:<p>Schizophrenia (SCZ) is a devastating psychiatric disorder. The aim of this study was to detect DNA methylation (5meC) profiles in whole blood associated with SCZ. The sample consisted of schizophrenia cases and controls selected from national population registers in Sweden. We employed MBD protein-based enrichment of the methylated DNA fraction, followed by single end sequencing (50 bp reads) on the SOLiD platform. To improve methylome-wide coverage we used an existing protocol variant, which increases the relative number of fragments from CpG poor regions. To analyze the data, we used an analysis pipeline described elsewhere7, that was designed specifically for MBD-seq and included alignment, quality control (QC) of reads and samples, coverage calculations for the 26,752,702 autosomal CpGs in the reference genome using a non-parametric estimate of the fragment size distribution, an in silico experiment to identify CpGs in loci with alignment problems, a 2-stage adaptive algorithm to combine data from correlated adjacent CpG sites in "blocks", principal component analysis (PCA), association testing, bioinformatics annotation and network analysis. After QC, 1459 subjects with on average 32.4 million high quality reads remained. Association testing was performed on 4.3 million high quality blocks after regressing out possible assay related technical artifacts, sex/age, and unmeasured confounders through the PCA components.</p>
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Project description:Agaricus bisporus is a soil-inhabiting fungus which is cultivated for production of white button mushrooms. A disease of A. bisporus has been previously described with a range of disease symptoms (yield loss, pinning delay, cap distortions and cap browning) which has been given collective name of “Mushroom Virus X” (MVX). The causes of this disease are not clear however prior to this research an association was found between the disease and double-stranded RNA molecules in the mushroom fruitbodies. The experiment was designed to examine causes and host responses of the disease causing the Brown Cap symptom in the cultivated mushroom A. bisporus. This microarray experiment was performed before the Agaricus bisporus genome was sequenced. The gene sequences used to design probes were from known and novel A. bisporus sequences and sequences of transcript fragments identified by Suppression Subtractive Hybridization of non-symptomatic and virus-diseased A. bisporus mushroom fruitbodies. The A. bisporus mushroom fruitbodies were grown on composted wheat straw using commercial cultivation procedures. The gene expression comparison was made of RNA isolated from 32 mushroom fruitbodies (Agaricus bisporus) samples: 20 samples from 5 separate virus-infected commercial mushroom farms with crops displaying the brown symptom (4 replicate samples per farm) and 12 samples from a non-infected crop grown at the University of Warwick. The precise composition of the viral load was the subject of this and future research/papers. Abstract of Manuscript submitted to Applied and Environmental Microbiology: Characterizing the viral agents causing brown cap mushroom disease of Agaricus bisporus by Daniel Eastwood, Julian Green, Helen Grogan, and Kerry Burton (Paper #AEM01093-15). The symptoms of viral infections of fungi range from cryptic to severe but there is little knowledge of the factors involved in this transition of fungal/viral interactions. Brown Cap Mushroom Disease of the cultivated Agaricus bisporus is economically important and represents a model system to describe this transition. Differentially expressed transcript fragments between mushrooms showing the symptoms of Brown Cap Mushroom Disease and control white non-infected mushrooms have been identified and sequenced. Ten of these RNA fragments have been found to be up-regulated over a thousand-fold between diseased and non-diseased tissue but are absent from the Agaricus bisporus genome sequence and hybridise to double-stranded RNA’s extracted from diseased tissue. We hypothesize these transcript fragments are viral and represent components of the disease-causing agent, a bipartite virus with similarities to the family Partitiviridae. The virus fragments were found at two distinct levels within infected mushrooms, at raised levels in infected, non-symptomatic, white coloured mushrooms and much greater levels (3,500-87,000 times greater) in infected mushrooms exhibiting brown colouration. In addition, differential screening revealed 9 up-regulated and 32 down-regulated host Agaricus bisporus transcripts. Chromametric analysis was able to distinguish colour differences between non-infected white mushrooms and white infected mushrooms at an early stage of mushroom growth. This method may be the basis for an ‘on-farm’ disease detection assay.
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