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ABSTRACT:
PROVIDER: PRJNA803973 | ENA |
REPOSITORIES: ENA
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Project description:Concurrent readout of sequence and base modifications from long unamplified DNA templates by PacBio single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
2024-05-01 | GSE245946 | GEO
Project description:Concurrent readout of sequence and base modifications from long unamplified DNA templates by PacBio single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
2022-08-08 | GSE210136 | GEO
Project description:Concurrent readout of sequence and base modifications from long unamplified DNA templates by PacBio single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
2024-05-01 | GSE225310 | GEO
Project description:Concurrent readout of sequence and base modifications from long unamplified DNA templates by PacBio single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
2024-05-01 | GSE225311 | GEO
Project description:We report the methylome sequencing and annotation of Burkholderia pseudomallei D286 based on high-throughput profiling using PacBio SMRT technology
2019-09-25 | GSE137930 | GEO
Project description:Aim: We aim to compare current (MeDIP-seq), new (Illumina Infinium 450K BeadChip) and future (PacBio) methods for whole genome DNA methylation analysis. As the interest in determination of disease methylation profiles increases, the scope, advantages and limitations of these methods requires assessment. There are key questions to answer and specific challenges to overcome. For example, how much detail/resolution is sufficient to identify regions of differential methylation and regions of biological/medical significance within a sample? How much coverage of the genome is required for accurate methylation analysis? Is it important to confirm which regions of the genome are unmethylated in addition to focusing on those that are methylated? Loss of methylation may be of equal importance within the cell since this may also contribute to disease pathogenesis. A multi-method (affinity enrichment/bisulphite-conversion based/direct sequencing of methyl-cytosine) and technology platform (Illumina HiSeq/PacBio/Illumina Infinium BeadChip) comparison will enable us to determine the strengths and weakness of each method. We propose to compare four methods using two DNA samples from the Coriell Institute for Cell Repository to assess both current and future capabilities for whole genome methylation analysis in parallel: A) MeDIP-seq using Illumina HiSeq B) Illumina Infinium HumanMethylation 450K BeadChip and C) whole genome methylation sequencing using PacBio. Existing single molecule deep bisulphite sequencing data generated previously from these same samples at the WTSI for targeted regions (30-40 genes) on the human X chromosome will be used to assess performance of each method. The methods selected for this study will generate data covering a range of resolutions from a whole genome scan to array (target defined) resolution and up to single base pair, single molecule resolution; the highest level of detail possible with methods currently available.Samples: DNA from sibling pair GM01240 (female) and GM01240 (male).Requirements: Both samples will be analysed using;A.MeDIP-seq using Illumina HiSeq (one HiSeq lane, 75bp paired end, per sample) B.Illumina Infinium HumanMethylation 450K BeadChipWe are expecting a potentially unnecessary high coverage using one HiSeq lane per sample. However, for the MeDIP procedure we do not have a multiplexing procedure in place. Our requirements for PacBio sequencing have been discussed with and will be supported by the Sequencing Technology Development group.
2013-01-22 | E-ERAD-139 | biostudies-arrayexpress
Project description:We report a method for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using non-specific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing using PacBio of these chromatin stencils enables nucleotide-resolution readout of the primary architecture of multi-kilobase chromatin fibers (Fiber-seq).
2020-06-25 | GSE146941 | GEO
Project description:We report a method for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using non-specific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing using PacBio of these chromatin stencils enables nucleotide-resolution readout of the primary architecture of multi-kilobase chromatin fibers (Fiber-seq).
2020-06-25 | GSE146940 | GEO
Project description:We report a method for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using non-specific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing using PacBio of these chromatin stencils enables nucleotide-resolution readout of the primary architecture of multi-kilobase chromatin fibers (Fiber-seq).
2020-06-25 | GSE146939 | GEO