The Experts below are selected from a list of 14169 Experts worldwide ranked by ideXlab platform

Timothy J Aitman - One of the best experts on this subject based on the ideXlab platform.

Matteo Pellegrini - One of the best experts on this subject based on the ideXlab platform.

  • Bisulfite Bolt: A Bisulfite Sequencing Analysis Platform
    2020
    Co-Authors: Colin Farrell, Michael A. Thompson, Anela Tosevska, Adewale Oyetunde, Matteo Pellegrini
    Abstract:

    Background: Bisulfite Sequencing is commonly employed to measure DNA methylation. Processing Bisulfite Sequencing data is often challenging due to the computational demands of mapping a low complexity, asymmetrical library and the lack of a unified processing toolset to produce an analysis ready methylation matrix from read alignments. To address these shortcomings, we have developed Bisulfite Bolt (BSBolt); a fast and scalable Bisulfite Sequencing analysis platform. Findings: We evaluated BSBolt against simulated and real Bisulfite Sequencing libraries. We found that BSBolt provides accurate and fast Bisulfite Sequencing alignments and methylation calls. We also compared BSBolt to several existing Bisulfite alignment tools and found BSBolt outperforms Bismark, BSSeeker2, BISCUIT, and BWA-Meth based on alignment accuracy and methylation calling accuracy. Conclusion:BSBolt offers streamlined processing of Bisulfite Sequencing data through an integrated toolset that offers support for simulation, alignment, methylation calling, and data aggregation. BSBolt is implemented as a python package and command line utility for flexibility when building informatics pipelines. BSBolt is available at https://github.com/NuttyLogic/BSBolt under a MIT license.

  • Bisulfite bolt a Bisulfite Sequencing analysis platform
    bioRxiv, 2020
    Co-Authors: Colin Farrell, Anela Tosevska, Adewale Oyetunde, M Thompson, Matteo Pellegrini
    Abstract:

    Background: Bisulfite Sequencing is commonly employed to measure DNA methylation. Processing Bisulfite Sequencing data is often challenging due to the computational demands of mapping a low complexity, asymmetrical library and the lack of a unified processing toolset to produce an analysis ready methylation matrix from read alignments. To address these shortcomings, we have developed Bisulfite Bolt (BSBolt); a fast and scalable Bisulfite Sequencing analysis platform. BSBolt performs a pre-alignment Sequencing read assessment step to improve efficiency when handling asymmetrical Bisulfite Sequencing libraries. Findings: We evaluated BSBolt against simulated and real Bisulfite Sequencing libraries. We found that BSBolt provides accurate and fast Bisulfite Sequencing alignments and methylation calls. We also compared BSBolt to several existing Bisulfite alignment tools and found BSBolt outperforms Bismark, BSSeeker2, BISCUIT, and BWA-Meth based on alignment accuracy and methylation calling accuracy. Conclusion: BSBolt offers streamlined processing of Bisulfite Sequencing data through an integrated toolset that offers support for simulation, alignment, methylation calling, and data aggregation. BSBolt is implemented as a python package and command line utility for flexibility when building informatics pipelines. BSBolt is available at https://github.com/NuttyLogic/BSBolt under an MIT license.

  • Targeted Bisulfite Sequencing for biomarker discovery
    Methods (San Diego Calif.), 2020
    Co-Authors: Marco Morselli, Colin Farrell, Liudmilla Rubbi, Heather L. Fehling, Rebecca Henkhaus, Matteo Pellegrini
    Abstract:

    Cytosine methylation is one of the best studied epigenetic modifications. In mammals, DNA methylation patterns vary among cells and is mainly found in the CpG context. DNA methylation is involved in important processes during development and differentiation and its dysregulation can lead to or is associated with diseases, such as cancer, loss-of-imprinting syndromes and neurological disorders. It has been also shown that DNA methylation at the cellular, tissue and organism level varies with age. To overcome the costs of Whole-Genome Bisulfite Sequencing, the gold standard method to detect 5-methylcytosines at a single base resolution, DNA methylation arrays have been developed and extensively used. This method allows one to assess the status of a fraction of the CpG sites present in the genome of an organism. In order to combine the relatively low cost of Methylation Arrays and digital signals of Bisulfite Sequencing, we developed a Targeted Bisulfite Sequencing method that can be applied to biomarker discovery for virtually any phenotype. Here we describe a comprehensive step-by-step protocol to build a DNA methylation-based epigenetic clock.

  • dna methylation estimation using methylation sensitive restriction enzyme Bisulfite Sequencing mrebs
    PLOS ONE, 2019
    Co-Authors: Giancarlo Bonora, Marco Morselli, Liudmilla Rubbi, Constantinos Chronis, Kathrin Plath, Matteo Pellegrini
    Abstract:

    Whole-genome Bisulfite Sequencing (WGBS) and reduced representation Bisulfite Sequencing (RRBS) are widely used for measuring DNA methylation levels on a genome-wide scale. Both methods have limitations: WGBS is expensive and prohibitive for most large-scale projects; RRBS only interrogates 6-12% of the CpGs in the human genome. Here, we introduce methylation-sensitive restriction enzyme Bisulfite Sequencing (MREBS) which has the reduced Sequencing requirements of RRBS, but significantly expands the coverage of CpG sites in the genome. We built a multiple regression model that combines the two features of MREBS: the Bisulfite conversion ratios of single cytosines (as in WGBS and RRBS) as well as the number of reads that cover each locus (as in MRE-seq). This combined approach allowed us to estimate differential methylation across 60% of the genome using read count data alone, and where counts were sufficiently high in both samples (about 1.5% of the genome), our estimates were significantly improved by the single CpG conversion information. We show that differential DNA methylation values based on MREBS data correlate well with those based on WGBS and RRBS. This newly developed technique combines the Sequencing cost of RRBS and DNA methylation estimates on a portion of the genome similar to WGBS, making it ideal for large-scale projects of mammalian genomes.

  • bs seeker2 a versatile aligning pipeline for Bisulfite Sequencing data
    BMC Genomics, 2013
    Co-Authors: Weilong Guo, Petko Fiziev, Weihong Yan, Shawn J Cokus, Xueguang Sun, Michael Q Zhang, Paoyang Chen, Matteo Pellegrini
    Abstract:

    DNA methylation is an important epigenetic modification involved in many biological processes. Bisulfite treatment coupled with high-throughput Sequencing provides an effective approach for studying genome-wide DNA methylation at base resolution. Libraries such as whole genome Bisulfite Sequencing (WGBS) and reduced represented Bisulfite Sequencing (RRBS) are widely used for generating DNA methylomes, demanding efficient and versatile tools for aligning Bisulfite Sequencing data. We have developed BS-Seeker2, an updated version of BS Seeker, as a full pipeline for mapping Bisulfite Sequencing data and generating DNA methylomes. BS-Seeker2 improves mappability over existing aligners by using local alignment. It can also map reads from RRBS library by building special indexes with improved efficiency and accuracy. Moreover, BS-Seeker2 provides additional function for filtering out reads with incomplete Bisulfite conversion, which is useful in minimizing the overestimation of DNA methylation levels. We also defined CGmap and ATCGmap file formats for full representations of DNA methylomes, as part of the outputs of BS-Seeker2 pipeline together with BAM and WIG files. Our evaluations on the performance show that BS-Seeker2 works efficiently and accurately for both WGBS data and RRBS data. BS-Seeker2 is freely available at http://pellegrini.mcdb.ucla.edu/BS_Seeker2/ and the Galaxy server.

Eun Joon Lee - One of the best experts on this subject based on the ideXlab platform.

  • Analyzing the cancer methylome through targeted Bisulfite Sequencing
    Cancer letters, 2012
    Co-Authors: Eun Joon Lee, Junfeng Luo, James Wilson, Huidong Shi
    Abstract:

    Bisulfite conversion of genomic DNA combined with next-generation Sequencing (NGS) has become a very effective approach for mapping the whole-genome and sub-genome wide DNA methylation landscapes. However, whole methylome shotgun Bisulfite Sequencing is still expensive and not suitable for analyzing large numbers of human cancer specimens. Recent advances in the development of targeted Bisulfite Sequencing approaches offer several attractive alternatives. The characteristics and applications of these methods are discussed in this review article. In addition, the bioinformatic tools that can be used for sequence capture probe design as well as downstream sequence analyses are also addressed.

  • targeted Bisulfite Sequencing by solution hybrid selection and massively parallel Sequencing
    Nucleic Acids Research, 2011
    Co-Authors: Eun Joon Lee, Lirong Pei, Gyan Srivastava, Trupti Joshi, Garima Kushwaha, Jeonghyeon Choi, Keith D Robertson, Xinguo Wang, John K Colbourne
    Abstract:

    We applied a solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput Bisulfite Sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of ~1 million CpGs in more than 21 408 CGIs and more than 15 946 transcriptional regulatory regions. Of the CpGs analyzed, 77–84% fell on or near capture probe sequences; 69–75% fell within CGIs. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. Differentially methylated regions (DMRs) were identified in the 5′-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X-chromosome among the three breast cancer cell lines analyzed. We chose 46 candidate loci (762 CpGs) for confirmation with PCR-based Bisulfite Sequencing and demonstrated excellent correlation between two data sets. Targeted Bisulfite Sequencing of three DNA methyltransferase (DNMT) knockout cell lines and the wild-type HCT116 colon cancer cell line revealed a significant decrease in CpG methylation for the DNMT1 knockout and DNMT1, 3B double knockout cell lines, but not in DNMT3B knockout cell line. We demonstrated the targeted Bisulfite Sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer epigenome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples.

  • Abstract 3003: Targeted Bisulfite Sequencing of CpG island DNA by solution hybrid selection and next-generation Sequencing
    Cellular and Molecular Biology, 2011
    Co-Authors: Eun Joon Lee, Lirong Pei, Gyan Srivastava, Garima Kushwaha, Jeonghyeon Choi, Xinguo Wang, Joshi Trupti, Kun Zhang, Huidong Shi
    Abstract:

    Recent studies using next-generation Sequencing have generated genome-wide, single-base resolution DNA methylation maps. However, it is still very costly to conduct whole genome shotgun Bisulfite Sequencing. Currently, few methods enable targeted Bisulfite Sequencing of CpG islands (CGIs) or other specific regions of interest in a highly flexible and efficient manner. Here we present a novel approach that combines solution-phase hybrid selection and massively parallel Bisulfite Sequencing to profile DNA methylation in targeted CGI and promoter regions. We designed 51,466 single strand DNA oligonucleotides (160-mer) which target 23,441 CGIs and the transcription start sites of 19,369 known genes in the human genome. The synthetic long DNA oligonucleotides were converted into biotinylated RNA probes for solution-phase hybridization capture of target DNA. The captured genomic DNA was treated with sodium Bisulfite, amplified by PCR and sequenced using Illumina GA IIx sequencer. Using this approach, we conducted Bisulfite Sequencing on captured DNA from three breast cancer cell lines, MCF10A, MCF7, and MDA-MB-231. 20-30 million single-end 75bp Sequencing reads were obtained for each cell line. The raw Sequencing reads were mapped to in silico Bisulfite-converted human genome. The methylation levels for CpG sites covered with at least 10 Sequencing reads were extracted, providing accurate quantification on 900,000-1,000,000 CpGs. 77-84% of CpGs analyzed in these samples fell on or near capture probe sequences; 69-75% lay properly on CGIs. One lane of Illumina Sequencing data was sufficient to determine the methylation status of over 22,000 CGIs (75% of all annotated CGIs) in the human genome. More than 85% of capture probes successfully yielded quantitative DNA methylation data of targeted regions. We chose 45 candidate loci (760 CpGs) for confirmation with PCR-based Bisulfite Sequencing and demonstrated excellent correlation between two data sets. This novel method was further validated by Sequencing three DNA methyltransferase (DNMT) knockout cell lines and the parental HCT116 colon cancer cell line. The targeted Bisulfite Sequencing data shows massive demethylation events in DNMT1 knockout (1KO) and double knockout (DKO) cell lines, but not in DNMT3B knockout (3BKO) and HCT116 cell lines. While the results indicate the critical role of DNMT1 in maintaining global DNA methylation, we have also identified DNMT3B specific demethylation patterns in a small group of genomic loci. In this study, we demonstrated the targeted Bisulfite Sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer genome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3003. doi:10.1158/1538-7445.AM2011-3003

Wolf Reik - One of the best experts on this subject based on the ideXlab platform.

  • single cell genome wide Bisulfite Sequencing for assessing epigenetic heterogeneity
    Nature Methods, 2014
    Co-Authors: Sebastien A Smallwood, Wolf Reik, Heba Saadeh, Felix Krueger, Simon Andrews, Christof Angermueller, Julian R Peat, Oliver Stegle
    Abstract:

    We report a single-cell Bisulfite Sequencing (scBS-seq) method that can be used to accurately measure DNA methylation at up to 48.4% of CpG sites. Embryonic stem cells grown in serum or in 2i medium displayed epigenetic heterogeneity, with '2i-like' cells present in serum culture. Integration of 12 individual mouse oocyte datasets largely recapitulated the whole DNA methylome, which makes scBS-seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.

  • Oxidative Bisulfite Sequencing of 5-methylcytosine and 5-hydroxymethylcytosine
    Nature Protocols, 2013
    Co-Authors: Michael J Booth, Neil M Bell, Miguel R Branco, Wolf Reik, Dario Beraldi, Shankar Balasubramanian
    Abstract:

    To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative Bisulfite Sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In Bisulfite Sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-seq, specific oxidation of 5hmC to 5-formylcytosine (5fC) and conversion of the newly formed 5fC to uracil (under Bisulfite conditions) means that 5hmC can be discriminated from 5mC. A positive readout of actual 5mC is gained from a single oxBS-seq run, and 5hmC levels are inferred by comparison with a BS-seq run. Here we describe an optimized second-generation protocol that can be completed in 2 d.

Gary C. Hon - One of the best experts on this subject based on the ideXlab platform.

  • Tet-Assisted Bisulfite Sequencing (TAB-seq)
    Methods in molecular biology (Clifton N.J.), 2017
    Co-Authors: Dali Han, Gary C. Hon
    Abstract:

    5-Hydroxymethylcytosine (5hmC) is a modified form of cytosine, which has recently been found in mammalian cells and tissues. 5hmC is derived from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) family protein-mediated oxidation and may regulate gene expression. Numerous affinity-based profiling methods have been developed to help understand the exact function of 5hmC in the genome. However, these methods have a relatively low resolution (~100 bp) without quantitative information of the modification percentage on each site. Here we demonstrated the detailed procedure of Tet-Assistant Bisulfite Sequencing (TAB-Seq), which can detect 5hmC at single-base resolution and quantify its abundance at each site. In this protocol, the genomic DNA is first treated with βGT and recombinant mTet1 consecutively to convert 5hmC to 5gmC and 5mC to 5caC, respectively. The treated genomic DNA can be directly applied to Bisulfite treatment to detect 5hmC on specific loci or applied to whole-genome Bisulfite Sequencing as needed.

  • Tet-assisted Bisulfite Sequencing of 5-hydroxymethylcytosine
    Nature protocols, 2012
    Co-Authors: Gary C. Hon, Chunxiao Song, Keith E. Szulwach, Peng Jin, Bing Ren
    Abstract:

    A complete understanding of the potential function of 5-hydroxymethylcytosine (5-hmC), a DNA cytosine modification in mammalian cells, requires an accurate single-base resolution Sequencing method. Here we describe a modified Bisulfite-Sequencing method, Tet-assisted Bisulfite Sequencing (TAB-seq), which can identify 5-hmC at single-base resolution, as well as determine its abundance at each modification site. This protocol involves β-glucosyltransferase (β-GT)-mediated protection of 5-hmC (glucosylation) and recombinant mouse Tet1(mTet1)-mediated oxidation of 5-methylcytosine (5-mC) to 5-carboxylcytosine (5-caC). After the subsequent Bisulfite treatment and PCR amplification, both cytosine and 5-caC (derived from 5-mC) are converted to thymine (T), whereas 5-hmC reads as C. The treated genomic DNA is suitable for both whole-genome and locus-specific Sequencing. The entire procedure (which does not include data analysis) can be completed in 14 d for whole-genome Sequencing or 7 d for locus-specific Sequencing.