Post-conversion targeted capture of modified cytosines in mammalian and plant genomes

Li, Qing; Suzuki, Masako; Wendt, Jennifer; Patterson, Nicole E.; Eichten, Steven R.; Hermanson, Peter J.; Green, Dawn; Jeddeloh, Jeffrey A.; Richmond, Todd; Rosenbaum, Heidi; Burgess, Daniel L.; Springer, Nathan M.; Greally, John M.

doi:10.1093/nar/gkv244

Cited by 64 publications

(73 citation statements)

References 57 publications

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“…For each standard, the correct quantitation of 5mC was retained through capture demonstrating that no quantitative bias is introduced, a critical metric that has not been previously reported for targeted-capture studies (Allum et al 2015;Ivanov et al 2013;Li et al 2015). For the low and high standards, quantitation differed between the WGBS, BOCS, and pyrosequencing data by <1 %, with the medium standard levels for the three methods differing slightly more (range = 6.6 %).…”

Section: Quantitative Accuracymentioning

confidence: 85%

“…This list of primary target regions was used for generation of capture probes. Tiled probes, ranging from 50 to 100 bp in length, were designed and synthesized by Roche Nimblegen against each strand and methylation state (Li et al 2015). The resulting capture set comprises a merged set of 22,146 primary target regions, with 4.2 million strand-specific probes covering 168,839,413 bp (6.6 million CG sites and over 73 million Cs in total).…”

Section: Probe Designmentioning

confidence: 99%

“…Analogous to exome sequencing, this approach would provide a good balance of genome coverage, necessary sequencing depth, absolute quantitation, and base-and strand-specific data. Methods which combine the power of bisulfite conversion with targeting to specific genomic locations have been developed, including oligonucleotide capture of target regions for bisulfite sequencing (Ivanov et al 2013;Li et al 2015). However, comprehensive capture sets of all promoters and CG islands in a genome and definitive demonstrations of capture enrichment, and most importantly, validation of quantitative accuracy have not been reported.…”

Section: Introductionmentioning

confidence: 99%

“…The demonstration of methylation state-independent capture and quantitative accuracy are critical as biasing of 5mC quantitation during capture enrichment is an obvious concern. Much like exome-capture sequencing, bisulfite oligonucleotide capture sequencing (BOCS), previously referred to as post-bisulfite conversion-targeted capture sequencing (Li et al 2015), reduces the amount of sequencing data required while still interrogating relevant portions of the genome making it a cost-effective approach for genomewide DNA methylation studies. BOCS entails generating a whole-genome bisulfite converted library followed by hybridization to sequence-specific probes, which capture targeted regions independent of methylation state.…”

Section: Introductionmentioning

confidence: 99%

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Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation

Masser

Stanford

Hadad

et al. 2016

AGE

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Epigenetic regulation through DNA methylation (5mC) plays an important role in development, aging, and a variety of diseases. Genome-wide studies of base-and strand-specific 5mC are limited by the extensive sequencing required. Targeting bisulfite sequencing to specific genomic regions through sequence AGE (2016) capture with complimentary oligonucleotide probes retains the advantages of bisulfite sequencing while focusing sequencing reads on regions of interest, enables analysis of more samples by decreasing the amount of sequence required per sample, and provides base-and strand-specific absolute quantitation of CG and non-CG methylation levels. As an example, an oligonucleotide capture set to interrogate 5mC levels in all rat RefSeq gene promoter regions (18,814) and CG islands, shores, and shelves (18,411) was generated. Validation using whole-genome methylation standards and biological samples demonstrates enrichment of the targeted regions and accurate base-specific quantitation of CG and non-CG methylation for both forward and reverse genomic strands. A total of 170 Mb of the rat genome is covered including 6.6 million CGs and over 67 million non-CG sites, while reducing the amount of sequencing required by~85 % as compared to existing wholegenome sequencing methods. This oligonucleotide capture targeting approach and quantitative validation workflow can also be applied to any genome of interest.

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Section: Quantitative Accuracymentioning

confidence: 85%

Section: Probe Designmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation

Masser

Stanford

Hadad

et al. 2016

AGE

View full text Add to dashboard Cite

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“…We term these approaches as bisulfite oligonucleotide capture sequencing (BOCS). Several very similar approaches have been developed for assessing the human (Wang et al 2011;Ivanov et al 2013;Allum et al 2015;Li et al 2015), mouse (Hing et al 2015;Li et al 2015), and rat (Masser et al 2016) genomes. In all of these approaches, capture of targeted regions is by complimentary oligonucleotide probes.…”

Section: Oligonucleotide Capturementioning

confidence: 99%

Analysis of DNA modifications in aging research

et al. 2018

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As geroscience research extends into the role of epigenetics in aging and age-related disease, researchers are being confronted with unfamiliar molecular techniques and data analysis methods that can be difficult to integrate into their work. In this review, we focus on the analysis of DNA modifications, namely cytosine methylation and hydroxymethylation, through nextgeneration sequencing methods. While older techniques for modification analysis performed relative quantitation across regions of the genome or examined average genome levels, these analyses lack the desired specificity, rigor, and genomic coverage to firmly establish the nature of genomic methylation patterns and their response to aging. With recent methodological advances, such as whole genome bisulfite sequencing (WGBS), bisulfite oligonucleotide capture sequencing (BOCS), and bisulfite amplicon sequencing (BSAS), cytosine modifications can now be readily analyzed with base-specific, absolute quantitation at both cytosine-guanine dinucleotide (CG) and non-CG sites throughout the genome or within specific regions of interest by next-generation sequencing. Additional advances, such as oxidative bisulfite conversion to differentiate methylation from hydroxymethylation and analysis of limited input/single-cells, have great promise for continuing to expand epigenomic capabilities. This review provides a background on DNA modifications, the current state-of-theart for sequencing methods, bioinformatics tools for converting these large data sets into biological insights, and perspectives on future directions for the field.

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Detection of somatic epigenetic variation in Norway spruce via targeted bisulfite sequencing

Heer

Ullrich

Hiß

et al. 2018

Ecology and Evolution

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Epigenetic mechanisms represent a possible mechanism for achieving a rapid response of long‐lived trees to changing environmental conditions. However, our knowledge on plant epigenetics is largely limited to a few model species. With increasing availability of genomic resources for many tree species, it is now possible to adopt approaches from model species that permit to obtain single‐base pair resolution data on methylation at a reasonable cost. Here, we used targeted bisulfite sequencing (TBS) to study methylation patterns in the conifer species Norway spruce (Picea abies). To circumvent the challenge of disentangling epigenetic and genetic differences, we focused on four clone pairs, where clone members were growing in different climatic conditions for 24 years. We targeted >26.000 genes using TBS and determined the performance and reproducibility of this approach. We characterized gene body methylation and compared methylation patterns between environments. We found highly comparable capture efficiency and coverage across libraries. Methylation levels were relatively constant across gene bodies, with 21.3 ± 0.3%, 11.0 ± 0.4% and 1.3 ± 0.2% in the CG, CHG, and CHH context, respectively. The variance in methylation profiles did not reveal consistent changes between environments, yet we could identify 334 differentially methylated positions (DMPs) between environments. This supports that changes in methylation patterns are a possible pathway for a plant to respond to environmental change. After this successful application of TBS in Norway spruce, we are confident that this approach can contribute to broaden our knowledge of methylation patterns in natural tree populations.

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Post-conversion targeted capture of modified cytosines in mammalian and plant genomes

Cited by 64 publications

References 57 publications

Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation

Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation

Analysis of DNA modifications in aging research

Detection of somatic epigenetic variation in Norway spruce via targeted bisulfite sequencing

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