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The most redundant sequences in human CpG island library are derived from mitochondrial genome.

ABSTRACT: An altered pattern of epigenetic modifications, such as DNA methylation and histone modification, is critical to many common human diseases, including cancer. Recently, mitochondrial DNA (mtDNA) was reported to be associated with tumorigenesis through epigenetic regulation of methylation patterns. One of the promising approaches to study DNA methylation and CpG islands (CGIs) is sequencing and analysis of clones derived from the physical library generated by methyl-CpG-binding domain proteins and restriction enzyme MseI. In this study, we observed that the most redundant sequences of 349 clones in a human CGI library were all generated from the human mitochondrial genome. Further analysis indicated that there was a 5,845-bp DNA transfer from mtDNA to chromosome 1, and all the clones should be the products of a 510-bp MseI fragment, which contained a putative CGI of 270 bp. The 510-bp fragment was annotated as part of cytochrome c oxidase subunit II (COXII), and phylogenetic analysis of homologous sequences containing COXII showed three DNA transfer events from mtDNA to nuclear genome, one of which underwent secondary transfer events between different chromosomes. These results may further our understanding of how the mtDNA regulates DNA methylation in the nucleus.

SUBMITTER: He X

PROVIDER: S-EPMC5054409 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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The most redundant sequences in human CpG island library are derived from mitochondrial genome.

He Ximiao X Tao Shu S Jin Jing J Hu Songnian S Yu Jun J

Genomics, proteomics & bioinformatics 20100601 2

An altered pattern of epigenetic modifications, such as DNA methylation and histone modification, is critical to many common human diseases, including cancer. Recently, mitochondrial DNA (mtDNA) was reported to be associated with tumorigenesis through epigenetic regulation of methylation patterns. One of the promising approaches to study DNA methylation and CpG islands (CGIs) is sequencing and analysis of clones derived from the physical library generated by methyl-CpG-binding domain proteins an ...[more]

PMID: 20691393

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Project description:BackgroundHybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors.ResultsWe used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses.ConclusionsWe first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

Project description:Core histone hyperacetylation, in particular of H4, is concentrated in the promoter-upstream regions of active genes and in certain cases is locuswide. Antibodies to hyperacetylated H4 were used to immunoprecipitate dinucleosomal chromatin derived from K562 human erythroleukemic cells by micrococcal nuclease digestion. The extracted DNA was made into a genomic library and was expected to contain sequences from genes active in K562 cells (an active, 'aDNA' library). Clones (180) were randomly selected from the library; 24 of 103 tested (23%) contained highly repeated sequences, as determined by their hybridization to total genomic DNA, and were not analyzed further. An additional 10 clones (6%) were shown to contain no insert DNA. The remaining 146 were sequenced and compared with the nucleic acid databases and in all six frames to the protein databases: Sixeen clones could be assigned to known genes, the majority of which (12) were tissue specific. All but 2 of these 16 corresponded to segments 5' of the coding sequences, as expected if H4 acetylation is concentrated at promoter regions. Thirty-three clones (23%) displayed high sequence identity to cDNAs in the expressed sequence tag database (dbEST). Northern blots and reverse transcription (RT)-PCR were used to determine the proportion of clones representing sequences expressed in K562 cells: Although only 1 of 34 tested clones showed a band in Northern hybridization, RT-PCR demonstrated that at least 12 of 40 tested clones (30%) were present in the mRNA population. Because a further 8 of these 40 clones were identified as gene fragments by database sequence comparisons, it follows that about half of this subset of 40 clones is derived from genes. The aDNA library is thus very gene rich and not skewed toward the most highly expressed sequences, as in mRNA libraries. The aDNA library is also rich in promoters and could be a valuable source of such sequences, particularly those that lack CpG islands or other features that allow their specific selection.

Dataset Information

The most redundant sequences in human CpG island library are derived from mitochondrial genome.

Publications

The most redundant sequences in human CpG island library are derived from mitochondrial genome.

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